Genetic investigation of the ubiquitin-protein ligase E3A gene as putative target in Angelman syndrome

Table 1 Clinical characteristics of patients with suspected Angelman syndrome

	Number of patients	Percentage (%)
Gender	33 male; 17 female
Hypotonia		100
Neck support	(32/50)	64
Walk without support	(21/50)	42
Sitting without support	(38/50)	76
Absent speech	(40/50)	80
Developmental delay	50/50)	100
Severe mental retardation	(50/50)	100
Microcephaly	(44/50)	88
Macrostomia	(40/50)	80
Clinical seizures	(44/50)	88
Occipital groove	(45/50)	90
Protruding tongue	(43/50)	86
Wide-spaced teeth	(35/50)	70
Prognathism	(40/50)	80
Unusually light hair or skin color	(13/50)	26
Easily provoked laughter	(50/50)	100
Hyperactivity	(48/50)	96
Gastro-esophageal reflux	(40/50)	80
Ataxic movements	(48/50)	96
Frequent drooling	(47/50)	94
History of sleep difficulties	(45/50)	90
Fascination with water	(40/50)	80
Autistic behavior	(14/50)	28

Table 2 Primer pairs used for single strand conformation polymorphism

Exon and primers	Forward and reverse primers (5’->3’)	Region	Annealing temperature (℃)
7
Ex7F	GCC ACC TGA TCT GAC CAC T	Intron	52
Ex7R	GCA GTC TAG GGC AAC TCA AA	Intron
8
Ex8AF	GCC TTG ATG ATA TGT TGA GC	Intron	55
Ex8AR	AAT TCT AGC GCC TTT CTT GT	Exon
Ex8BF	GCC TGC ACG AAT GAG TTT TGT	Exon	55
Ex8BR	AGT TAT TAT TCC TGT CCG TTA CC	Intron
9
Ex9AF	TGT TTG GCT GTT TTA CTT TTA GA	Intron	55
Ex9AR	GGC ATC AAT ATC CAC AGA CAC A	Exon
Ex9BF	AGA AGC ATC TTC CTC AAG G	Exon	55
Ex9BR	CAC TTC CCC TCC CAC TAC	Exon
Ex9CF	CAA TGA ATT TAA CAG TCG A	Exon	55
Ex9CR	CAT CAT CTA TGA TAT GGT CAC G	Exon
Ex9DF	CGC ATG TAC AGT GAA CGA AGA A	Exon	55
Ex9DR	TGC ACA GGA ACA ACA AAA GTA T	Intron
10
Ex10F	GTT TGC TTT CTG TTT CCA TTT AC	Intron	52
Ex10R	ATC CTT CTT TTG CTG CTC TTC	Intron
11
Ex11F	CAA TGT TGC ATG CCT AAT TAC A	Intron
	GGT ACT TCG GTC AGA TTA AAA C	Intron
12
Ex12F	GGG GAC TGG AGG GAT ACT GT	Intron	55
	ACA TGC TTT GAA AGT GTT AAT G	Intron
13
Ex13F	GAA ATT GTT AAG AAG TAG GTG	Intron	52
Ex13R	ATA TGT CTT AGT TAT CTG CTA	Intron
14
Ex14F	AGG TGT CTG CAA AAA GTC	Intron	55
Ex14R	TTA GCT CTG AAA AAT GGT G	Intron
15
Ex15F	ATA ATG AAT GCC AAA CTG AA	Intron	55
Ex15R	ATA TGT ATG TGA CGA GGA ATG	Intron
16
Ex16F	CCC ATG ACT TAC AGT TTT CCT G	Intron	55
Ex16R	AAG AAG GGA GGC ACA GAC AT	Intron

Table 3 Ubiquitin-protein ligase E3A polymorphisms detected in our group of clinically diagnosed Angelman syndrome patients. The nucleotide and codon positions refer to the complete ubiquitin-protein ligase E3A cDNA sequence

Four common polymorphisms	New polymorphisms
c.2064+9T>C → rs79328837 (intro13)	c.2220+14T>C (intron14)
c.1713A>G → RS34670662 (exon 11)	c.30-47_30-46 insT (Exon7)
c.2221-40_2221-38delGTA, RS149854051 (intron 14)	c.2507+43T>A (Exon15)
c.486A>T; p.Ala162 =, RS28528079 (exon9A)

Table 4 Identified exonic variants in the first consanguineous family

Gene (gene)	SHPRH	SLC30A9	HBS1L	TAAR6	TAAR2	SASH1	LOC100287896	PCF11	ANKRD42	PDGFD	DIXDC1
Chromosome	6	4	6	6	6	6	11	11	11	11	11
Location (varLocation)	Exonic	Intronic	intronic	Exonic	Exonic	Exonic	Exonic	Exotic	Exonic	Exonic	Exonic
Effect (codingEffect)	Nonsynonymous			Nonsynonymous	Nonsynonymous	Nonsynonymous	Nonsynonymous	Nonsynonymous	Nonsynonymous	Nonsynonymous	Nonsynonymous
cDNA (cNomen)	c.4331C>T	c.528-7T>C	c.2043+5T>G	c.865C>T	c.467C>T	c.1126C>T	c.4C>T	c.3355C>T	c.676A>G	c.7C>G	c.226G>A
Protein (pNomen)	p.A1444V			p.P289S	p.T156I	p.P376S	p.R2C	p.H1119Y [Histidine (His)- Tyrosine (Tyr)]	p.N226D [Asparagine (Asn)- Aspartic Acid (Asp)]	p.R3G [Arginine (Arg)- Glycine (Gly)]	p.G76S [Glycine (Gly)- Serine (Ser)]
Pathogenicity	09/11	This mutation probably has no impact on splicing	Activation of an intronic cryptic donor site.	07/11	10/11	09/11		08/11	09/11	03/Nov	0/11
			Potential alteration of splicing
GME Variome (%)	Not available	Not available	Not available	0.1	Not available	Not available	Not available	0.1	0.1	0.1	Not available
Gnomad browser (%)	0.0008	0.04	0.2	0.006	0.001	0.001	0.4	0.2	0.3	0.01	0.002

GME: Greater middle east.

Table 5 Identified exonic variants in the second consanguineous family

Gene (gene)	KMT5A	KMT5A	STK36	PIK3CB	GPR149	RARRES1	KPNA4	NOS1	CAMKK2	WDR66	SBNO1
Chromosome	12	12	2	3	3	3	3	12	12	12	12
Location (varLocation)	Exonic	Exonic	Exonic	Exonic	Exonic	Exonic	Exonic	Exonic	Exonic	Exonic	Intronic
Effect (codingEffect)	Non-synonymous	Non-synonymous	Non-synonymous	Non-synonymous	Non-synonymous	Non-synonymous	Non-synonymous	Non-synonymous	Non-synonymous	Non-synonymous	Non-synonymous
cDNA (cNomen)	c.904>C	c.995>C	c.2516 A	c.2150>G	c.1404A>C	c.230C>T	c.1103>G	c.1922>T	c.1612_1614dupAAA	c.196_197insAGAAAGAGGAGGAGG	c.3220+5C>G
Protein (pNomen)	p.C302R	p.L332P	p.R839Q	p.N717S	p.R468S	p.P77L	p.N368S	p.A641V	p.K538dup	p.E65_G66insEKEEE	No significant splicing motif alteration detected. This mutation probably has no impact on splicing
Pathogenicity	03/11	10/11	06/11	01/11	01/11	10/11	03/11	09/11	01/11	01/11	Not available
GME Variome (%)	Not available	Not available	0.05	0.9	0.1	Not available	Not available	Not available	Not available	Not available	0.01
Gnomad browser (%)	Not available	Not available	0.7	0.1	0.04	0.004	0.003	Not available	12	Not available	0.01

GME: Greater middle east.

Table 6 Gene expression in both Family 1 and Family 2

Gene	Function
Gene expression in Family 1
TAAR2	Relevance to brain function and behavior, including schizophrenia, depression, anxiety, and drug addiction
CITED2	Gene is identified in human endothelial cells and neonatal brain. It is implicated in the development of the heart and neural tube
HPRH	Ubiquitously expressed protein that contains motifs characteristics of several DNA repair proteins, transcription factors, and helicases. A possible candidate for the tumor suppressor gene
SASH1	Highly expressed in brain, heart, lung, ovary, and kidney. It is also identified as a candidate tumor suppressor gene in breast cancer
DIXDC1	Highly expressed in the brain and in specific brain regions; important in embryonic cortical development
SLC30A9	Shows ubiquitous expression in various human tissues, with high expression in the fetal brain, cerebellum, skeletal muscle, and kidney. Sub-cellular localization studies suggested that it is expressed in the vesicular cytosolic compartment, possibly in the endoplasmic reticulum
Gene expression in Family 2
KMT5A	Is a lysine methyl-transferase that predominantly mono-methylates lysine-20 (K20) of histone H4
SETD8	Influences transcriptional regulation, heterochromatin formation, genomic stability, cell cycle progression, and development
STK36	Important for brain development
PIK3CB	Plays a role in systemic insulin-like growth factor (IGF1) regulation and human longevity
RARRES1	Implicated in hyperproliferation, inflammatory skin diseases and, prostate cancer
GPR149	Increases fertility in mice, and causes prostatic cancer
NOS1	Important for the brain and peripheral nervous system
SBNO1	Implicated in cognition and psychoses, essential roles in vertebrate brain development