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 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: 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