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P. Bianchi and E. Fermo
ing the impact of genotype in clinical trials (see the section “From genotype to new therapies”).
Deep intronic variants
An increasing number of pathogenic deep intronic mutations has been described across different disease con- ditions,53 and these mutations have been considered to jus- tify the number of PK-deficient patients in whom it is not possible to find molecular defects. Among the 278 partic- ipants initially enrolled in the PKD NHS, 21 were consid- ered ineligible for the study because of the inability to demonstrate two pathogenic variants even after excluding the large deletion analyzed by long-range polymerase chain reaction.34,35 This aspect has important implications, obviously from the diagnostic point of view, but also with regards to the possibility to access new specific therapies, as further discussed in the next paragraphs.26,54 Laboratory
confirmation of deep intronic variants is often difficult and may require specific testing such as loss of heterozygosity by analyzing an allele-specific cDNA, or the more com- plex minigene construct approach.55
In a recent study a deep intronic mutation (c.283+109C>T in intron 2) was detected by whole exome sequencing in compound heterozygosity with the mis- sense mutation p.G332S, and was considered responsible for creating an alternative splicesome by in silico analysis; rapid mRNA degradation was confirmed by the observa- tion of loss of heterozygosity of the p.G332S variant at the cDNA level.56
In another study, 13 PK-deficient related individuals with one or no pathogenic variants identified in the PKLR gene were analyzed by whole exome sequencing or whole genome sequencing. Five patients had an alterna- tive diagnosis with mutations in GATA1, KIF23, and
Table 2. Most common mutations of the PKLR gene, ethnic distribution and allelic frequency.
Mutation Effect
c.1529G>A p.R510Q
c.1456C>T p.R486W c.1468C>T p.R490W c.721G>T p.E241* c.994G>A p.G332S c.992 A>G p.D331G
Pathogenic variants with strong ethnic association
Exon Mutation type
11 Missense
11 Missense 11 Missense 7 Nonsense 8 Missense 8 Missense
Geographical distribution/ethnicity
Nothern EU, USA
Southern EU, India Japan Caucasian Caucasian India
Pennsylvania Amish, Indian
African Roma community Vietnamese
rs
rs113403872
rs116100695 rs200133000 rs201953584 rs773626254 rs1443439423
rs118204085
rs147689373 na
na
Allelic frequency Ref. (gnomAD Exome)
0.000358 34
0.00305 34,36 0.000127 37 0.0000485 16 0.0000557 16 0.00000398 36
0.000127 36,38
0.000694 39 na 40 na 17,41
c.1436G>A
c.829G>A c.1437-518_1618+440del c.283+1914_1434del
p.R479H/abnormal splicing
p.E277K 1149 bp deletion 5006 bp deletion
10
7 i10-i11 i3-10
Missense/splicing
Missense Large deletion Large deletion
Figure 3. DNA sequence of the erythroid-specific PKLR promoter region. Conserved sequences between human and rat PKR promoter are underlined, the black arrow indi- cates the PK-R transcriptional start site. Yellow boxes indicate the GATA-1 motif, the green boxes identify the CAC/Sp1 motif and the blue box identifies the PKR-RE regulatory ele- ment. Colored arrows indicate motif direction. Mutated nucleotides reported in the litera- ture associated with pyruvate kinase deficiency are indicated in red and reported in more detail in the box.
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haematologica | 2020; 105(9)