PRKN Variant Annotation And Functional Assay Evidence

PRKN Variant Annotation Table, Continuation

Table headers:

Index
Databases
Parkin Variant
Annotation with clinical evidence
Functional group
Annotation with clinical and functional evidence

Continuation rows:

index	databases	Parkin variant	clinical evidence annotation	functional group	clinical + functional annotation
18	Disease and population	p.Q34R	Benign	3	Benign
19	Disease and population	p.AS2E / p.A52E?	Benign	4	Benign
20	Disease and population	p.S167N	Benign	4	Benign
21	Disease and population	p.R334C	Benign	5	Benign
22	Disease and population	p.V380L	Benign	4	Benign
23	Disease and population	p.D394N	Benign	4	Benign
24	Disease and population	p.D18N	Uncertain significance	4	Likely benign
25	Disease and population	p.R33Q	Uncertain significance	4	Likely benign
26	Disease and population	p.P37L	Uncertain significance	5	Likely benign
27	Disease and population	p.R42C	Uncertain significance	4	Likely benign
28	Disease and population	p.R42H	Uncertain significance	4	Likely benign
29	Disease	p.W54R	Uncertain significance	3	Uncertain significance
30	Disease and population	p.R104W	Uncertain significance	3	Uncertain significance
31	Disease	p.K161N	Uncertain significance	2	Pathogenic
32	Disease	p.M192V	Uncertain significance	4	Uncertain significance
33	Population	p.V224A	Uncertain significance	5	Likely benign
34	Disease and population	p.R234Q	Uncertain significance	5	Likely benign
35	Disease and population	p.T240M	Uncertain significance	2	Pathogenic
36	Population	p.V248I	Uncertain significance	4	Likely benign
37	Disease	p.C253F	Uncertain significance	1	Pathogenic
38	Disease and population	p.R256C	Uncertain significance	5	Likely benign
39	Disease and population	p.D280N	Uncertain significance	4	Likely benign
40	Disease	p.C289G	Uncertain significance	1	Pathogenic
41	Disease and population	p.E310R	Uncertain significance	4	Likely benign
42	Disease	p.G328E	Uncertain significance	4	Uncertain significance
43	Disease and population	p.R334H	Uncertain significance	4	Uncertain significance
44	Disease	p.T351P	Uncertain significance	4	Uncertain significance
45	Disease and population	p.G359D	Uncertain significance	3	Uncertain significance
46	Disease and population	p.R366W	Uncertain significance	4	Likely benign
47	Disease and population	p.R396G	Uncertain significance	4	Uncertain significance
48	Disease	p.C418R	Uncertain significance	1	Pathogenic
49	Disease and population	p.P437L	Uncertain significance	2	Pathogenic
50	Disease and population	p.E444Q	Uncertain significance	4	Uncertain significance
51	Disease and population	p.M458L	Uncertain significance	5	Likely benign

Figure 1: Parkin Missense Variants Functional Alterations

Caption and legend:

Figure 1. Parkin missense variants displayed a wide range of functional alterations.
 
A
Disease specific database (Patients)
24
51
164
Population database
 
2 Pathogenic
1 Likely pathogenic
21 Uncertain significance
 
8 Pathogenic (P)
2 Likely pathogenic (Lp)
4 Likely benign (Lb)
6 Benign (B)
31 Uncertain significance (Us)
 
164 Uncertain significance
 
B
Patients only
Patients and population
Normalized function (%)
Mitophagy
GFP intensity

Note below Figure 1:

위 그림 fig1에서, mitophagy는 under CCCP (4H),
but GFP intensity 는 (steady-state) parkin protein level (without CCCP)

Parkin Variant Functional Assay Table

Table headers:

column	visible header
domain/category	leftmost column, partially clipped
variant	Parkin variant
inclusion	Inclusion formation
protein expression	Parkin protein expression level (GFP intensity) fig1b
mitophagy	Mitophagy
example	Example

Method row:

measurement	visible method text
Parkin protein expression level	Transfection of U2OS with WT or GFP-Parkin (24h) -> flow cytometry -> FACS (U2OS cells express very low levels of Parkin, 그러니 GFP signal 은 다 exogenous 임)
Mitophagy	Transfection of U2OS by transfecting plasmid DNA using jetPRIME (Polyplus) -> the cells will stably express mtKeima -> selection with G418 for 2 weeks and sorting using flow cytometry -> mtKeima is a pH-sensitive fluorescent protein that is targeted to mitochondria and exhibits a large shift in its emission wavelength upon en-gulfment in the acidic compartment of lysosomes (ie keima goes into mito and its wavelength is different in lysosome, 즉 얼마나 많이 lysosome 내로 갔는지 판단가능), in the presence of CCCP (20 uM, 4h)

Variant rows:

domain/category	Parkin variant	inclusion formation	protein expression	mitophagy	example / note
Ubl domain	p.R42P (ARG42PRO)	X	down arrow	down arrow
Ubl domain	p.V56E	X	down arrow	down arrow
	p.K211N		equals	down arrow
RORBR domain	p.C212Y	O	down arrow	down arrow
RORBR domain	p.C238W (= p.Cystein238Tryptophan)	O	down arrow	down arrow	(Zanellati, 2015 #712)
RORBR domain	p.C253Y	O	down arrow	down arrow
	p.G284R		equals	down arrow
	p.T415N		equals	down arrow
	p.C431F		equals	down arrow
RORBR domain	p.C441R	O	down arrow	down arrow
	p.T240R		equals	down arrow
RORBR domain	p.R275W (= p.Arg275Tryptophan) = R>W, C.823>T, or rs34424986, VCV000007050	O	down arrow	note present	FATHMM/COSMIC/Yi notes
clipped left note	R275Q: p.Arg275Gln; c.824G>A; Variation ID: 931482; Accession: VCV000931482.2; rs 번호는 없네			circle marker
	p.G430D (= p.Gly430Asp)		equals	down arrow	(Mortiboys, 2008 #719)
	p.K161N		note present
	p.C253F		note present

Notes embedded in the functional-assay table:

p.R275W (= p.Arg275Tryptophan)
= R>W, C.823>T, or rs34424986,
VCV000007050 (이걸로 UNBBN에서 여럿 보이나 다 정상)
 
FATHMM prediction Pathogenic score: score 0.87
(이건 cancer 용인가?) is with
https://cancer.sanger.ac.uk/cosmic/mutation/overview?id=98884592
 
Yi 2019 의 suppl table 보면 (excel), benign point from MAF in population 이 1임,
이건 normal people 에서도 일부 발견된다는 말 아닌가?
그러나 Benign points from homozygotes in population 가 0인걸 보면,
적어도 homoz는 일반에서 안 발견되나 봄.
 
R275Q: p.Arg275Gln
c.824G>A Variation ID: 931482, Accession: VCV000931482.2,
rs 번호는 없네
 
Js: these two were not found as clinical pathogenic, but from functional assays
(mitophagy, protein expression), these two look pathogenic

ACMG-AMP Clinical Evidence Criteria Note

Text:

Note) [methods] 이 전체가 ACMG-AMP criteria
 
1. Clinical evidence (from public database) 먼저:
Pathogenic vs Benign (scoring system, algorithm-based) (2019 Yi)

Table:

pathogenic	benign
the more patients in the family homoz or compound heteroz in Parkin ('Segregation within family', the more pathogenic	Age > 50
suppl table 보면 (excel), 13개 pathogenic variants 는 모두 Benign points from homozygotes in population 가 0임. 즉 거꾸로 0인 것들만 모아서 pathogic 이라고 했다는 뜻.	The more common in population database (ExAC, Exosome variant serve, dbSNP), the more benign, (even if they are homoz!)

Bottom clipped fragment:

... 해서 13 pathogenic & likely pathogenic variants 정리
2.

Uncertain Spans

location	text/status	reason
statusbar/body overlap	75?, Yi's study: 13, Yi's study: 10, 70% of all mutations	Some text is visible through the Word status/status overlap and should not be used as structured data without checking the image.
row 19	p.AS2E / p.A52E?	Variant characters are visually ambiguous.