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 (2N)

Patient: 32 yo, male

Variant: VWF NM_000552.5 c:2527C>T, p.(Arg760Cys), exon 17, noted to be dominant effect

Note: Proband also has an Arg91Gln mutation in exon 20 of VWF too.

Phenotypes: Mild bleeding history, epistaxis, bleeding gums, bleeding after tooth extraction. 11 years old had severe hemorrhage following tonsillectomy. Previous diagnosis of hemophilia A.

Family: father has history of epistaxis but never eamined prior to study. Father has the Arg760Cys mutation at heterozygous level. Mother is asymptomatic and classified as hemophilia A carrier following hemophilia A diagnosis in son. Mother also has the Arg91Gln mutation in exon 20 at heterozygous level.

Molecular Phenotypes: mild decrease in plasma FVIII and VWF levels, normal platelet VWF content, decreased FVIII binding capacity of VWF. Causes retention of pro-VWF(suggest defects in the maturation process of VWF). Abnormal VWF and platelet GPIb interaction(documented by impaired or absent RIPA and decreased VWF:RCo).

Disease: Von-willebrand Disorder Type 2B, Noncanonical

Patient: 55 YO, Italian male

Note: these heterozygous variants are in cis mutations

Variant1: VWF NM_000552.5 c.2771G>A p.(R924Q), in exon 21, D'D3 domain affected

Variant2: VWF NM_000552.5 c.6532G>T p.(A2178S), in exon 37, D4 domain affected

Phenotypes: Mucosal and cutaneous bleeding, low VWF antigent and VWF activity, mild thrombocytopenia, increased ristocetin-induced platelet aggregation, deficiency in high-molecular-weight multimers. Epistaxis, repeated GI bleeding, easy bruising, ISTH BAT score of 7.

Note: Proband initially diagnosed with VWD type 1 in 2010 but later re-diagnosed as VWD-Type 2.

Family: No reported family history of bleeding, parents unable to be included in family work-up as they were deceased by time of study. Paternity test was performed for family, parental link established. Patient's daughter does not present the same mutation noted in proband but has heterozygous polymorphic variant inherited from mother which is: VWF NM_000552.5 c.3379C>T p.(P1127S), in exon 25. Daughter did not show menorrhagia or hemorrhagic disorder except for large hematoma on the thigh after trauma of moderate intensity.

Molecular workup: Paternity test, Sanger-sequencing to validate genetic variants, multimer analysis with electrophoresis, platelet aggregation analysis with Ristocetin, electron micrographs to observe conformation differences.

Prediction workup: Structural analysis with I-TASSER modeling program to identify perturbed structure. A2178S is predicted as tolerated in all in-silico systems used.

Database Information: Effect of the R924Q mutation is noted in literature database but has contradicting reports of effects altering VWF levels. Some studies do report it as a polymorphic variant

Variant is present in dbSNP database (rs33978901). MAF in European population 0.01< MAF< 0.02

Effect of A2178S variant is present in dbSNP database (rs34230288), MAF = 0.02 in European population.

Disease: Myopathic Ehlers-Danlos Syndrome (mEDS)

Patient(s): 47 yo male, japanese descent

Variant: COL12A1 NM_004370.6: c.395-1G>A (Homozygous variant, at splice acceptor site in exon 6, causes in-frame skipping)(located in the genomic region encoding the first von Willebrand factor A domain)

Family: consanguineous parents with no related features of mEDS, healthy older brother

Phenotypes (Childhood): hypotonia, weak spontaneous movements, scoliosis, torticollis, soft plams, undescended testes, motor developmental delay, slender build, triangular face, short palpebral fissures, small nose, small mouth, large ears, bilateral knee dislocations, short stature.

Phenotypes (Adulthood): short stature, high palate, hypermobile small joints, deformed cervical spine, brachycephaly, bilateral long deformed 5th finger, severe scoliosis post surgical fixation, asymmetric pelvis

Classification: sequencing panel found the variant and it was confirmed through sanger sequencing.

According to ClinGenSVI recommendation: PVS1_Strong

Applicable criteria:

1) the one at a GT-AG 1,2 splice site

2) the one exerting exon skipping or use of a crypic splice site that preserved the reading frame.

3) the one at a truncated/altered region critical to protein function; was classified as PM3_Supporting (homozygous)

Variant not registered in gnomAD (PM2_Supporting)

Partial defect of first vWA in collagen XII judged as PS3

Disease: Von Willebrand Disease (VWD)

Patient(s): Found in 2 families

Variant: VWF NM_000552.5: c.2311A>G, p.(M771V Homozygous variant in exon 18 (VWF D' domain; 8 residues down from proteolytic VWFpp furin cleavage site)

Family: In family 1 there are 4 homozygous patients (2 male and 2 female), and one heterozygous patient (1 female). The affected females are denoted as person 1 and person 4 and the affected males are person 2 and person 3. There are three WT family members (1 female and 2 male), grandparents of these members are of unknown genotype including a daughter of an affected female and a WT male. Note here that in the family a p.R2663P variant has co-segregated with the above-mentioned variant but is not suspected to be the pathogenic driver of resulting bleeding tendency.

In family 2 the parents of the homozygous affected male are of unknown genotype. The affected male is denoted as person 7.

Phenotypes: Person 1- nose bleed, skin bleed, GI bleeding, oral cavity bleeds, Menorrhagia, muscle bleeding, and joint bleeding. Receives on-demand treatment for bleeding.

Person 2-Nose bleed, skin bleed, bleeding from small wounds, oral cavity bleeds, bleeding after tooth extraction, joint bleeding. Received prophylactic treatment, reduced to on-demand treatment after a few years.

Person 3-Nose bleed, skin bleed, oral cavity bleeds, bleeding after tooth extraction, muscle bleeding. Receives on-demand treatment for bleeding phenotype.

Person 4- Nose bleed, bleeding from small wounds, oral cavity bleed, bleeding after tooth extraction, joint bleeding. Received prophylactic treatment that was increased after her menarche.

Person 7- Nose bleed, oral cavity bleeds, bleeding after surgery or trauma, joint bleeding. Previously on prophylaxis, now managing bleeding with on-demand treatment.

Note that both the p.R2663P co-segregated variant and p.M771V variant are reported in NCBI dbSNP database but functional effect not yet established.

NGS confirmed the genotype of all study participants.

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: Platelet-type Von-willebrand Disorder (PT-VWD)

Patient: 17 yo, male, adopted

Variant: GP1BA NM_000173.7: c:580C>T p.(P.Leu194Phe), Heterozygous, gain-of-function

Phenotypes: moderate bleeding phenotype, ISTH-BAT bleeding score of 3, recurrent epistaxis, easy bruising, mild thrombocytopenia

Family: Adopted, no other family history mentioned, segregation studies not performed.

Genetic analysis performed: found variant in GP1BA, results obtained by sanger sequencing.

Variant present in gnomAD(rs368111193): low allele frequency, contradictory classifications

Variant is not present in ClinVar, LOVD, or HGMD databases

According to this paper, ACMG guidelines classified this variant as a VUS.

This paper entered it into Clinvar (var ID 1693270)

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

Patient 2 Variant(s):

VWF NM_000552.5: c.4135C>T p.(Arg1379Cys) Exon 28 VWF NM_000552.5: c.4130C>T p.(Ala1377Val) Exon 28 VWF NM_000552.5: c.3797C>T p.(Pro1266Leu) Exon 28 VWF NM_000552.5: c.3835G>A p.(Val1279Ile) Exon 28

Note: Pro1266Leu and Val1279Ile are in trans with Ala1377Val and Arg1379Cys

Note2: MAF of Ala1377Val is present in Exome Variant Server (<0.01) and 1000 Genomes database (<0.02), designated as rare variant, typically found in indiv with African ethnicity

Family: Relatives molecular analysis showed Arg1379 and Ala1377Val variants mentioned above were in cis

Patient 2 Phenotype: Mild bleeding symptom Nearly normal VWF:Ag value, reduced VWF:RCo value slight loss of HMWM with smear decreased/slightly decreased proteolysis Slightly reduced RIPA VWFpp/VWF:Ag ratio shows increased VWF clearance VWF platelet levels reduced reduced rBpIba binding in VWF plasma VWF:GPIbM values slightly increased

Note: Treated with desmopressin in case of minor surgeries or delivery

Note 2: Slightly decreased RIPA may be explained by presence of 2B New York Variant (Pro1266Leu) that mitigates RIPA assay in 2M phenotype.

In silico analysis available:

I-Mutant 3.0 states decrease in A1 domain stability for both mutations (Ala1377Val = -0.91, Arg1379Cys = -1.36)

PYMOL predicts Ala1377Val does not alter formation of hydrogen bonds with Arg1374 residue and water. Predicts 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

Ala1377Val was previously reported in 3 other patients Publications:

Millar CM, Riddel AF, Mellors G, Yee TT. The spectrum of VWD type 2 phenotypes associated with A1 domain mutations posters. J Thromb Haemost 2009; 7: 531–2.

Logsdon BA, Dai JY, Auer PL et al. A variational Bayes discrete mixture test for rare variant association. Genet Epidemiol 2014; 38: 21–30.

Final note: Authors suggest patients' 2M phenotype is due to the presence of Ala1377Val and Arg1379Cys together to create synergistic effect. Though difficult to discern with this specific patient having two other variants in addition to the two mentioned above.

In at least one other published version the first two items on this list are not peacock fans and Japanese screens but “ormolu garden gates, handleless cups”. The Oxford Book of Victorian Ghost Stories, ed. Michael Cox and R.A. Gilbert

Ormolu is the gilding technique of applying finely ground, high-carat gold–mercury amalgam to an object of bronze, and for objects finished in this way. The mercury is driven off in a kiln leaving behind a gold coating. The French refer to this technique as "bronze doré"; in English, it is known as "gilt bronze". https://en.wikipedia.org/wiki/Ormolu?wprov=sfti1

In at least one other published version the first two items on this list are not peacock fans and Japanese screens but “ormolu garden gates, handleless cups”. The Oxford Book of Victorian Ghost Stories, ed. Michael Cox and R.A. Gilbert

Ormolu is the gilding technique of applying finely ground, high-carat gold–mercury amalgam to an object of bronze, and for objects finished in this way. The mercury is driven off in a kiln leaving behind a gold coating. The French refer to this technique as "bronze doré"; in English, it is known as "gilt bronze". https://en.wikipedia.org/wiki/Ormolu?wprov=sfti1

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Sandra Ciesek. (2021, December 8). Unsere ersten Daten zur Neutralisation von Omicron versus Delta sind fertig: 2x Biontech, 2x Moderna, 1xAZ/1x Biontech nach 6 Monaten 0% Neutralisation bei Omicron, auch 3x Biontech 3 Monate nach Booster nur 25% NT versus 95% bei Delta. Bis zu 37fache Reduktion Delta vs. Omicron https://t.co/w0gHww26sg [Tweet]. @CiesekSandra. https://twitter.com/CiesekSandra/status/1468465347519041539

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ReconfigBehSci. (2021, December 14). RT @thehowie: "...The vaccine provided 70 percent protection in fully vaccinated individuals against severe complications that would requir… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1470749072160133128

How are new Covid cases impacting sport? (n.d.). BBC Sport. Retrieved December 23, 2021, from https://www.bbc.com/sport/59682938

ReconfigBehSci. (2021, December 14). RT @thehowie: BREAKING South Africa: 82% increase in cases, week over week. 34.9% positive rate. Record 7-day average. Not near platea… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1470814593610047490

Dr Nisreen Alwan 🌻. (2021, December 23). We’re in unchartered territory. Https://t.co/O1GGcXpMll [Tweet]. @Dr2NisreenAlwan. https://twitter.com/Dr2NisreenAlwan/status/1474051397570248716

Kai Kupferschmidt. (2021, December 10). “the severity profile of Omicron cases must be interpreted along with an understanding of its capacity to re-infect (and infect the vaccinated)“ This is what I have been trying to explain the last few days. As usual @nataliexdean does it better (and in color)! [Tweet]. @kakape. https://twitter.com/kakape/status/1469270407995867139

Yaniv Erlich. (2021, December 8). Updated table of Omicron neuts studies with @Pfizer results (which did the worst job in terms of reporting raw data). Strong discrepancy between studies with live vs pseudo. Https://t.co/InQuWMAm4l [Tweet]. @erlichya. https://twitter.com/erlichya/status/1468580675007795204

ReconfigBehSci. (2021, December 12). RT @ryan_landay: > A new diverse genome has appeared within the B.1.1.529 lineage that has all of the shared mutations of B.1.1.529, some o… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1470066521615605766

ReconfigBehSci. (2021, December 9). RT @RachelLavin: As Omicron reality dawns, consider investing in better masks: "....Tight-fitting FFP2 masks provide 75 times better pro… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1474030898551087113

Campbell, D., Sabbagh, D., & Devlin, H. (2022, January 7). Military deployed at London hospitals due to Omicron staff shortages. The Guardian. https://www.theguardian.com/world/2022/jan/07/military-deployed-at-london-hospitals-due-to-omicron-staff-shortages

ReconfigBehSci. (2021, November 26). RT @mvankerkhove: Following the meeting of the @WHO TAG-VE today, WHO classifies B.1.1.529 as a variant of concern named Omicron. We call… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1464302372100976650

ReconfigBehSci. (2021, December 12). RT @EricTopol: If you think Prior Covid protects against Omicron (B.1.1.529), think again https://medrxiv.org/content/10.1101/2021.12.08.21267491v1 and "Neutralizing antibo… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1470434736304803845

ReconfigBehSci. (2021, November 26). vaccine equity has been a disaster, but I do wonder whether the exclusive focus on donations does the US/EU comparison justice. The EU allowed the export of huge numbers of EU produced doses at a time when the US did not (and EU itself was struggling to meet demand). [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1464255575416520749

Karan, A. (2022). We cannot afford to repeat these four pandemic mistakes. BMJ, 376, o631. https://doi.org/10.1136/bmj.o631

ReconfigBehSci on Twitter: “RT @BNODesk: BREAKING: U.S. reports 716,714 new coronavirus cases, setting world record for cases in one day” / Twitter. (n.d.). Retrieved January 7, 2022, from https://twitter.com/SciBeh/status/1478987541332348933

ReconfigBehSci. (2021, December 23). Risk of hospital stay 40% lower with Omicron than Delta, UK data suggests https://t.co/VupN8hY4qq [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1473916654421643266

ReconfigBehSci. (2021, November 26). maybe this is a good moment to remind people that makers of mRNA vaccines have been extensively prepping for the possibility of new variants. Biontech/Pfizer have given a timeline of 100 days to the delivery of a retooled version of their vaccine [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1464225264523747359

ReconfigBehSci. (2021, December 11). RT @MackayIM: Booster access brought forward to slow spread of Omicron COVID-19 variant https://t.co/PukbPydylw [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1469633384511119364

Eric Topol. (2022, January 2). As the world turns.....vertical the Omicron signature (|) @OurWorldInData https://t.co/LOBiaiBapD [Tweet]. @EricTopol. https://twitter.com/EricTopol/status/1477433315896430592

Kissane, E. (2021, December 23). We’re About to Lose Track of the Pandemic. The Atlantic. https://www.theatlantic.com/ideas/archive/2021/12/were-about-to-lose-track-of-the-pandemic/621097/

ReconfigBehSci. (2021, December 13). RT @DrEricDing: 11) Omicron is doubling every 1.6 days in UK 🇬🇧 according to @DrWilliamKu’s figure. That’s way faster growth than in South… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1470451594378502154

Prof. Christina Pagel. (2021, December 23). UKHSA surveillance report shows reinfections rising sharply as Omicron takes hold https://t.co/UGArY4sbOA [Tweet]. @chrischirp. https://twitter.com/chrischirp/status/1474041921249189896

Thomas, T., & Duncan, P. (2021, December 23). If Omicron is the dominant variant in UK, why is the number of confirmed cases so low? The Guardian. https://www.theguardian.com/world/2021/dec/23/if-omicron-is-the-dominant-variant-in-uk-why-is-the-number-of-confirmed-cases-so-low

ReconfigBehSci. (2021, December 13). Important update on the now withdrawn @BallouxFrancois tweet identifying hopeful turning of the corner in SA omicron data [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1470311016114364420

ReconfigBehSci. (2021, December 11). RT @DrEricDing: DOUBLING % in one day: The spike of probable #Omicron variant in Scotland🏴󠁧󠁢󠁳󠁣󠁴󠁿 has critically surged—Now at 13.3%, up fro… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1469596523222781954

ReconfigBehSci. (2021, December 8). RT @kallmemeg: NEW: @UKHSA Mini Omicron Update Omicron VOC-21NOV-01 (B.1.1.529) update on cases, S gene target failure and risk assessment… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1468673329494216726

John Burn-Murdoch. (2021, November 25). Five quick tweets on the new variant B.1.1.529 Caveat first: Data here is very preliminary, so everything could change. Nonetheless, better safe than sorry. 1) Based on the data we have, this variant is out-competing others far faster than Beta and even Delta did 🚩🚩 https://t.co/R2Ac4e4N6s [Tweet]. @jburnmurdoch. https://twitter.com/jburnmurdoch/status/1463956686075580421

CNN, I. K. (n.d.). Vaccine inequity and hesitancy made the Omicron variant more likely, scientists say. CNN. Retrieved December 23, 2021, from https://www.cnn.com/2021/11/28/world/omicron-coronavirus-variant-vaccine-inequity-intl-cmd/index.html

ReconfigBehSci. (2021, December 22). RT @chrischirp: Positivity rising steeply too—There are definitely many more people we aren’t catching https://t.co/4xDPwYQtrx [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1473695678610214927

ReconfigBehSci on Twitter: “RT @chrischirp: As well as Tom’s new one (B.1.1.529), C.1.2 seems to be spreading in S Africa—C.1.2 was the one with lots of worrying mut…” / Twitter. (n.d.). Retrieved December 23, 2021, from https://twitter.com/SciBeh/status/1463597407082532864

ReconfigBehSci. (2021, December 16). RT @MadsAlbertsen85: #Omicron update from Denmark. Omicron cases on the 12th of December adjusted up to 20.5% (+2%). Omicron is now having… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1471516083819462670

ReconfigBehSci. (2021, December 11). RT @thehowie: BREAKING: Hospitalizations South Africa Hospitalizations⬆️67% week over week. Gauteng Province⬆️66%. GP⬆️1.6% from yesterd… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1469685234149072897

ReconfigBehSci. (2021, December 9). a rather worrying development- a (local) newspaper “fact checking” the new German health minister simply by interviewing a virologist who happens to have a different view. There’s simply no established “fact” as to the severity of omicron in children at this point in time [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1469037817481334786

Trevor Bedford. (2021, December 11). We find that logistic growth of Omicron sequence fraction looks similar between the UK, the US and Germany with roughly 1% of sequenced cases in all three countries being Omicron on Dec 1. 3/10 https://t.co/De0t2xreU9 [Tweet]. @trvrb. https://twitter.com/trvrb/status/1469784757261127685

Gollub, R. L. (2022). Brain changes after COVID revealed by imaging. Nature. https://doi.org/10.1038/d41586-022-00503-x

ReconfigBehSci. (2021, December 17). RT @EricTopol: No evidence that Omicron is less severe, new @imperialcollege study reports https://ft.com/content/020534b3-5a54-4517-9fd1-167a5db50786 https://t.co/5YYzJ6tVtc [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1472156681098469380