. 2021 Jan 12;11:764. doi: 10.1038/s41598-020-80755-7

Table 1.

Number of affected family members, in silico pathogenicity predictions and ACMG classification for novel variants found in the cohort.

Gene	Patient #	Affected family members	Nucleotide change	Affected exon(s)/intron(s)	Amino acid alteration	gnomAD MAF [%]	Pathogenicity predictions			Splice predictions	ACMG
Gene	Patient #	Affected family members	Nucleotide change	Affected exon(s)/intron(s)	Amino acid alteration	gnomAD MAF [%]	CADD	REVEL	M-CAP	Splice predictions	Classification	Criteria
*FBN1*^c	1	1	c.(?_1317)_(1837 + 1_1838-1)del	Upstream of exon 1 and exons 1–15	p.?	NA	NA	NA	NA	Not done	LP^#	PVS1, PM2
	3	1 (de novo)^a	c.1130G > A	Exon 10	p.(Cys377Tyr)	Absent	33	0.927	0.915	Not done	LP	PM1, PM2, PM5, PM6, PP2, PP3, PP5
	4	2	c.1463G > A	Exon 12	p.(Cys488Thr)	Absent	32	0.937	0.892	Not done	LP	PM1, PM2, PM5, PP2, PP3, PP5
	5^b	2	c.(1468 + 1_1469-1)_(1837 + 1_1838-1)del	Exons 13–15	p.?	NA	NA	NA	NA	Not done	P^#	PVS1, PM2, PS4
	6^b	3	c.(1468 + 1_1469-1)_(1837 + 1_1838-1)del	Exons 13–15	p.?	NA	NA	NA	NA	Not done	P^#	PVS1, PM2, PS4, PP1
	8	2	c.1867T > A	Exon 16	p.(Cys623Ser)	Absent	27.6	0.771	0.493	Not done	LP	PM1, PM2, PM5, PP2, PP3
	9	2	c.2419 + 3delinsTTTTAGATCCATATTTTAG	Intron 20	p.?	Absent	14.96	NA	NA	Impact on splicing	VUS^#	PM2, PP3
	14	1 (de novo)^a	c.3589 + 1G > A	Intron 29	p.?	Absent	34	NA	NA	Impact on splicing	P	PVS1, PM2, PM6, PP3
	16	1	c.3635G > A	Exon 30	p.(Cys1212Tyr)	Absent	32	0.965	0.899	Not done	LP	PM1, PM2, PM5, PP2, PP3
	18	2	c.4491C > G	Exon 37	p.(Cys1497Trp)	Absent	17.95	0.768	0.820	Not done	LP	PM1, PM2, PM5, PP2, PP3
	21	2	c.4817-1_4819delGATA	Intron 39/exon 40	p.?	Absent	37	NA	NA	Impact on splicing	P	PVS1, PM2, PP3
	22	1 (de novo)^a	c.5467_5474dupGAATGCAT	Exon 45	p.(Ile1825Metfs*71)	Absent	33	NA	NA	Not done	P	PVS1, PM2, PM6, PP3
	25	3	c.5621G > T	Exon 46	p.(Cys1874Phe)	Absent	33	0.991	0.981	Not done	LP	PM1, PM2, PP1, PP2, PP3
	27	1 (de novo)^a	c.5671 + 1G > C	Intron 46	p.?	Absent	33	NA	NA	Impact on splicing	P	PVS1, PM2, PM6, PP3
	28	1 (de novo)^a	c.5917 + 1G > T	Intron 48	p.?	Absent	34	NA	NA	Impact on splicing	P	PVS1, PM2, PM6, PP3, PP5
	29	1	c.5966G > C	Exon 49	p.(Cys1989Ser)	Absent	25.2	0.818	0.946	Not done	LP	PM1, PM2, PM5, PP2, PP3
	30	3	c.5993G > T	Exon 49	p.(Cys1998Phe)	Absent	33	0.956	0.928	Not done	LP	PM1, PM2, PM5, PP1, PP2, PP3
	32	2	c.(7204 + 1_7205-1)_(7819 + 1_7820-1)del	Exons 59–63	p.?	NA	NA	NA	NA	Not done	LP^#	PVS1, PM2
	34	2	c.7817T > A	Exon 63	p.(Val2606Asp)	Absent	33	0.665	0.270	Not done	LP	PM1, PM2, PP2, PP3
*SKI*^d	38	1 (de novo)^a	c.104C > G	Exon 1	p.(Pro35Arg)	Absent	23.6	0.759	0.964	Not done	LP	PM1, PM2, PM5, PM6, PP3
*TGFBR2*^e	43	1	c.1453C > A	Exon 6	p.(Arg485Ser)	Absent	29.4	0.868	0.374	Not done	LP	PM1, PM2, PM5, PP2, PP3
*TGFBR2*^e	44	1 (de novo)^a	c.1454G > C	Exon 6	p.(Arg485Pro)	Absent	33	0.937	0.427	Not done	LP	PM1, PM2, PM5, PM6, PP2, PP3

The functional impact of the identified variants was predicted by the Combined Annotation Dependent Depletion (CADD) tool, the Rare Exome Variant Ensemble Learner (REVEL) scoring system, and the Mendelian Clinically Applicable Pathogenicity (M-CAP) Score. CADD is a framework that integrates multiple annotations in one metric by contrasting variants that survived natural selection with simulated mutations. Reported CADD scores are phred-like rank scores based on the rank of that variant’s score among all possible single nucleotide variants of hg19, with 10 corresponding to the top 10%, 20 at the top 1%, and 30 at the top 0.1%. The larger the score the more likely the variant has deleterious effects; the score range observed here is strongly supportive of pathogenicity, with all observed variants ranking above ~ 99% of all variants in a typical genome and scoring similarly to variants reported in ClinVar as pathogenic (~ 85% of which score > 15)⁶¹. REVEL is an ensemble method predicting the pathogenicity of missense variants with a strength for distinguishing pathogenic from rare neutral variants with a score ranging from 0 to 1. The higher the score the more likely the variant is pathogenic⁶². M-CAP is a classifier for rare missense variants in the human genome, which combines previous pathogenicity scores (including SIFT, Polyphen-2, and CADD), amino acid conservation features and computed scores trained on mutations linked to Mendelian diseases. The recommended pathogenicity threshold is > 0.025⁶³. Splice site prediction scores were calculated for wild-type and mutated sequences by using the programs Human Splicing Finder 3.1, NetGene2, and the Berkeley Drosophila Genome Project Database^65–68. Genetic tolerance at the affected amino acid position in the protein was predicted by MetaDome⁶⁴. All variants were classified according to the guidelines of the American College of Medical Genetics (ACMG) either by use of an adjusted automated interpretation by VarSome (https://varsome.com/)⁶⁰or in case of whole exon deletions and Indels (#) by manual application of the guidelines.

LP likely pathogenic, NA not applicable, P pathogenic, VUS variant of unclear significance.

^aPaternity not confirmed.

^bApparently non-consanguineous families.

^cFBN1 mRNA reference number: NM_000138.4.

^dSKI mRNA reference number: NM_003036.3.

^eTGFBR2 mRNA reference number: NM_001024847.2.