Table 3.

Molecular diagnostic findings from iNeurons RNA-seq

ID	Sex	Age	Gene	Isoform	Variant DNA	Zygosity	Inheritance	Variant RNA	RNA consequence	Detection algorithma	TPM iNeuron median	TPM fibroblast median	iN required?b
1	M	4 years	ITPR1	NM_001378452.1	c.5980−17G>A	het	de novo	r.5979_5980ins[5980−15_5980−1/5979+1_5980−18,a,5980−16_5980−1]	two events: 15 nt intron inclusion at splice acceptor; entire intron inclusion	AS	6.03	1.16	yes
2	M	22 years	DCX	NM_001195553.2	c.946+4588G>T	hem	de novo	r.946_947ins[946+4619_947−1]	13,549 nt intron inclusion at splice acceptor	AS	2.31	0	yes
3	F	4 years	MBD5	NM_001378120.1	c.−925+35307_−557+13765delchr2:148056991_148356101del	het	maternal	r.−924_−558del	5′ UTR exons 2–4 deletion containing 337 nt	AE (0.84), AS	3.32	2.99	yes
4	F	7 years	CACNA1A	NM_001127222.2	c.5015G>C	het	de novo	r.5015g>c (r.632_784del)	skewed variant allele expression at 88% fraction; skipping of exon 5 (153 nt); uncertain if the two events are related	MAE; AS	6.48	2.54	yes
5	M	36 years	POLR3A	NM_007055.4	c.1771−7C>G	het	paternal	r.1771_1909del/1643_1909del	two events: skipping of exon 14 (139 nt); skipping of exons 13–14 (267 nt)	AS (moderate effect)	13.92	10.76	no
					c.3892−297_3892−221del	het	maternal	r.3891_3892ins[3892−1_3892−227/3892−74_3892−227]	two events: 227 nt intron inclusion at splice acceptor; 154 nt cryptic exon inclusion from intron 29	AS (moderate effect)
6	F	5 years	PIEZO2	NM_001378183.1	c.5257−1G>A	het	maternal	r.5258del	1 nt shift of splice acceptor	AE (0.17), AS (misalignment, NMD), MAE	4.56	2.14	yes
					c.1528−1G>A	het	paternal	r.1528del	1 nt shift of splice acceptor	AE (0.17), AS (misalignment, NMD), MAE
7	M	2 years	LZTR1	NM_006767.4	c.2178C>A	het	paternal	r.2178c>a	nonsense	not applicable	25.41	36.41	no
					c.1943−256C>T	het	maternal	r.1942_1943ins[1942+342_1943−262/1942+360_1943−262]	two events: 117 nt cryptic exon inclusion; 99 nt cryptic exon inclusion	AE (0.53), AS
8	F	4 years	USP9X	NM_001039591.3	c.1315−284G>T	mosaic	de novo	r.1314_1315ins[1315−281_1315−176/1315−281_1315−172]	two events: 106 nt or 110 nt cryptic exon inclusion	AE (0.36), AS	43.08	42.06	no
9	F	16 years	BRAF	NM_001374258.1	seq[GRCh38] t(5; 7)(q31.3; q34) NC_000005.10:g.140964619_qterdelins[NC_000007.14:g.140799141_qter] NC_000007.14:g.140799137_qterdelins[NC_000005.10:g.140964621qter]	het	de novo	r.-226_980:(?)/−226_980:980_981ins[980+1_980+1225]:(?)	two (or more) events: fusion gene of 5′ BRAF with sequence from 7q34; fusion gene of 5′ BRAF plus 1225 nt intron retention with sequence from 7q34; the question mark denotes uncertainty of the breakpoints	AE (0.66), AS	6.35	5.83	no
10	M	2 years	TMEM161B	NM_153354.5	c.800+5G>A	het	maternal	r.660_800del	skipping of exon 8 (141 nt)	AS (candidate disease-associated gene)	5.29	4.06	no
					c.980T>C	het	paternal	r.980u>c	missense	not applicable
11	M	26 years	NSD2	NM_001042424.3	NC_000004.12:g.1869269_1873124del	het	unknown	(NSD2)x53%	expression reduction of the entire transcript	AE (0.53)	7.70	13.85	no
12	F	4 years	POGZ	NM_015100.4	c.2546−20T>A	het	de novo	r.2546_2570del/2545_2571ins[2545+1_2546−21,a,2546−19_2571−1]	two events: skipping of exon 18 (25 nt); retention of introns 17–18 (446 nt)	AS	20.87	22.03	no
13	M	10 years	MYCBP2	NM_015057.5	c.8005C>T	het	de novo	(MYCBP2)x75%	stopgain variant likely causes NMD and skewed allele fraction	AE (0.75), MAE	16.59	14.33	no
14	F	2 years	VARS1	NM_006295.3	c.3288G>T	het	paternal	r.3288delins[u,3288+1_3289−1]	entire intron 27 retention (71 nt)	AE (0.35), AS	30.3	38.98	no
					c.2590_2592delAGCinsTGA	het	maternal	(VARS1)x35%	stopgain variant likely causes NMD and skewed allele fraction	AE (0.35), MAE
15	F	10 years	TIAM1	NM_001353694.2	c.1996−78G>A, c.1585−5707G>T, c.−188−13607G>A	het	unknown	(TIAM1)x2%	expression reduction of the entire transcript	AE (0.02)	2.63	2.55	no
16	F	3 years	NAV2	NM_145117.5	c.5011_5012del	het	maternal	(NAV2)x25%	frameshift variants likely cause NMD	AE (0.25)	3.04	8.67	no
					c.6580del	het	paternal
17	M	2 years	RBM28	NM_018077.3	c.1745G>A	het	maternal	r.1714_1788del	variant likely destroys exon splicing enhancer and cause skipping of exon 16 (75 nt)	AE(0.73), AS	5.26	5.37	no
					c.1489_1492dup	het	paternal	(RBM28)x73%	frameshift variants likely causes NMD	AE(0.73), MAE
18	F	9 years	FBN1	NM_000138.5	c.248−151A>G	het	paternal	r.247_248ins[248–282_248−152]	inclusion of cryptic exon (131 nt) with premature stop codon likely causing NMD	AE (0.58), AS (low abundance due to NMD), MAE	524.4	517.6	no

Abbreviations: AE, abnormal expression; AS, abnormal splicing; MAE, mono-allelic expression; NMD, nonsense-mediated decay.

The reduction of TIAM1 expression is considered as a possible molecular diagnosis with weaker evidence compared to all other variants in this table. This is because of a lack of the matching DNA variant underlying the expression change. Three rare variants in TIAM1 are listed. However, their causal relationship with the expression reduction is uncertain.

The FBN1 variant is considered as a partial molecular diagnosis, which may partly explain the Marfanoid phenotype in this individual, but not the neurological phenotypes.

The POLR3A variants are considered as a possible molecular diagnosis because of the atypical phenotypic match.

TMEM161B⁵⁴ and MYCBP2⁵⁵ are recently identified disease-associated genes not included in disease-associated gene databases such as OMIM at the time of the analysis. Therefore, the findings in these two individuals are considered investigational.

The phenotypes of each proband are summarized as Human Phenotype Ontology (HPO) terms (Data S2).

^aContent in parentheses following AE indicates the expression fold change in this individual; the content in parentheses following AS indicates that the splicing event escaped detection from the analysis pipeline but was rescued by manual analysis with special considerations.

^bThe iNeuron process is required to activate the gene or isoform of interest to facilitate variant interpretation.