Clinical characteristics of patients
In the present study, 17 families were included (18 females and 17 males, ages, 13–68 years and with a mean age of 33.29 ± 9.46 years). The RCH sex ratio, female: the male was nearly 1:1.
CHB was observed in 14 cases of RCH (40%); PHEO and KC in 2 cases (5.7%), and PC was reported in 1 patient (2.8%) (Table 2). Among the 17 families who were investigated for VHL mutations, 13 families (76.5%) had at least one family member with a VHL variant while 4 families had no familial history (23.5%).
Table 2. Germline VHL mutation and its phenotypes in 17 RCH families
Number
|
Family
|
Sex/Age
|
FHx
|
Exon
|
Variants
|
AA change
|
VHL type
|
RCH
|
CHB
|
PCC
|
PC
|
KC
|
ACMG classification
|
Mutational event (type)
|
1
|
1
|
F/13
|
Yes
|
3
|
Del
|
-
|
1
|
+
|
+
|
-
|
-
|
-
|
-
|
Deletion
|
2
|
F/48
|
3
|
Del
|
|
1
|
+
|
-
|
-
|
-
|
-
|
-
|
Deletion
|
3
|
2
|
F/33
|
Yes
|
2 & 3
|
Del
|
-
|
1
|
+
|
+
|
-
|
-
|
-
|
-
|
Deletion
|
4
|
M/30
|
3
|
Del
|
-
|
1
|
+
|
-
|
-
|
-
|
-
|
-
|
Deletion
|
5
|
M/26
|
3
|
Del
|
-
|
1
|
+
|
-
|
-
|
-
|
-
|
-
|
Deletion
|
6
|
M/68
|
2
|
Del
|
-
|
1
|
+
|
+
|
-
|
-
|
-
|
-
|
Deletion
|
7
|
3
|
M/35
|
No
|
Last base of exon 2
|
c.463G > T
|
V155L
|
1
|
+
|
+
|
-
|
-
|
-
|
Pathogenic
|
Splice
|
8
|
4
|
M/33
|
Yes
|
Last base of exon 2
|
c.463G > A
|
V155M
|
1
|
+
|
+
|
-
|
-
|
-
|
Pathogenic
|
Splice
|
9
|
5
|
F/30
|
Yes
|
IVS2; canonical
splice donor for exon 2
|
c.463 + 3A > G
|
-
|
1
|
+
|
+
|
-
|
-
|
-
|
Likely Pathogenic
|
Splice
|
10
|
6
|
M/32
|
No
|
2
|
Del
|
-
|
1
|
+
|
-
|
-
|
-
|
-
|
-
|
Deletion
|
11
|
7
|
M/27
|
Yes
|
1
|
c.208G > A
|
E70K
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
12
|
F/61
|
1
|
c.208G > A
|
E70K
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
13
|
8
|
F/31
|
Yes
|
3
|
c.563T > G
|
L188R
|
1
|
+
|
+
|
-
|
-
|
+
|
Pathogenic
|
Missense
|
14
|
F/25
|
3
|
c.563T > G
|
L188R
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
15
|
F/17
|
3
|
c.563T > G
|
L188R
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
16
|
M/31
|
3
|
c.563T > G
|
L188R
|
1
|
+
|
+
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
17
|
M/54
|
3
|
c.563T > G
|
L188R
|
1
|
+
|
+
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
18
|
9
|
M/41
|
Yes
|
1
|
c.208G > A
|
E70K
|
1
|
+
|
+
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
19
|
10
|
M/28
|
Yes
|
3
|
c.467A > G
|
Y156C
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
20
|
M/26
|
3
|
c.467A > G
|
Y156C
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
21
|
M/16
|
3
|
c.467A > G
|
Y156C
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
22
|
M/14
|
3
|
c.467A > G
|
Y156C
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
23
|
F/38
|
3
|
c.467A > G
|
Y156C
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
24
|
F/23
|
3
|
c.467A > G
|
Y156C
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
25
|
11
|
F/35
|
Yes
|
1
|
Del
|
-
|
2A
|
+
|
-
|
+
|
-
|
-
|
-
|
Deletion
|
26
|
F/39
|
3
|
c.499C > T
|
R167W
|
2A
|
+
|
+
|
+
|
-
|
-
|
Pathogenic
|
Missense
|
27
|
12
|
M/32
|
Yes
|
2
|
Del
|
-
|
1
|
+
|
+
|
-
|
-
|
-
|
-
|
Deletion
|
28
|
13
|
F/29
|
No
|
1
|
C.208G > A
|
E70K
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
29
|
14
|
F/45
|
Yes
|
3
|
c.508G > C
|
V170L
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
30
|
F/67
|
3
|
c.508G > C
|
V170L
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
31
|
15
|
M/25
|
Yes
|
3
|
Del
|
-
|
1
|
+
|
+
|
-
|
-
|
-
|
-
|
Deletion
|
32
|
F/47
|
3
|
Del
|
-
|
1
|
+
|
+
|
-
|
-
|
+
|
-
|
Deletion
|
33
|
16
|
M/36
|
No
|
3
|
c.376_382del
|
D126Ffs*31
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Frameshift
|
34
|
17
|
F/28
|
Yes
|
1
|
c.208G > A
|
E70K
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
35
|
F/48
|
1
|
c.208G > A
|
E70K
|
1
|
+
|
-
|
-
|
-
|
-
|
Pathogenic
|
Missense
|
Mutation distribution in RCH patients with other symptoms of VHL
In this study, 10 different mostly pathogenic VHL germline mutations were identified in 17 families (Table 2). Analysis of sequences of 17 families represented missense mutations were the most common (20/35, 57.1%), followed by large/complete deletions (11/35, 31.4%), splice site variants (3/35, 8.6%) and frameshift (1/35, 2.8%) (Table 2). Amino acid substations were located in 2 domains, α and β, while there was no mutation in the codons from 1 to 69 (Fig. 2). Individuals carrying amino acid substitutions in α and β -domain were 17 cases and 7 cases, respectively. Six deletions, especially occurring at α- domain and 4 deletions concentrated in β- domain and 1 deletion were located in 2 domains.
The frequency of missense mutations was in exon 1 (17.1%) and exon 3 (40%), the large/complete deletion was in exon 1(2.8%), 2 (11.4%) and 3 (20%) (Table 3). The 2 splice site variants of missense changes were placed in the last base of exon 2 (5.7%) and another was placed in the canonical splice donor for exon 2 (2.8%), and there was 2.8% frameshift in exon 2. Eight males and 12 females had missense mutations, 6 males and 5 females had large/complete deletions, 2 males and 1 female had splice site variants, and 1 male had frameshift mutations.
Table 3. Distribution of genetic mutations in the VHL gene in RCH patients
Overall, the most common variants involved in exon 3 locate α- domain of pVHL. Interestingly, the most frequently observed mutations in our patients were c.208G > A (p.E70K) which occurred in 4 families (23.5%). However, mutations c.563T > G (L188R) and c.467A > G (Y156C) were presented with the highest incidence in 8th and 10th family.
Based on ACMG classification, 23 patients had pathogenic criteria and 1 patient had likely pathogenic criteria.
Genotype-phenotype correlation
Six individuals carrying the c.208G > A (p.E70K) mutation showed RCH or CHB; 5 patients had only RCH and the other patient manifested RCH and CHB. Eighth family carrying the c.563T > G (p. L188R) missense mutation manifested RCH, CHB or KC; 2 patient had only RCH and the other patient manifested RCH, CHB and KC. Tenth family carrying the c.467A > G (Y156C) missense mutation manifested only RCH. Six patients carrying the large/complete deletion manifested RCH and CHB, 4 patients had only RCH and 1 patient manifested RCH and PCC (Table 1).
The pathogenic missense mutations c.208G > A (p.E70K) were placed within the HIF-α binding site (residues 65–117) and 2 splice site variants were in Chaperonin-containing tailless complex polypeptide 1 (CCT) (residues 100– 155), where VHL needs to be coupled correctly with elongin BC (Fig. 2) [17, 18]. PCC was found in 2 subjects in the family number 11; Proband was carrying a heterozygous deletion in the exon 1. Her sister has a different mutation (a missense mutation c.499C > T (p.R167W) in the exon 3) with more severe clinical manifestations and a poor prognosis. The pedigree of the investigated this family was shown in Fig. 1.
With a comparison between these two groups RCH + VHL + and RCH + VHL-, the following results were obtained (Table 3):
Statistically significant different was not observed between the age distribution of RCH + Variant + and RCH + variant – that were 32.7 ± 6.42 and 34.5 ± 11.90, respectively (P = 0.501). Also, there was not a statistically significant difference in the sex distribution between RCH + Variant - and RCH + variant - (P = 0.603) (Table 4).
Table 4. Distribution of age and sex in RCH patients.
Twenty-four RCH patients carried amino acid substitutions and deletions located in α domain and 12 RCH patients carried amino acid substitutions and deletions located in β –domain, 68.6% vs. 34.3%, respectively.
In addition, it was shown that 11 CHB in RCH patients carried amino acid substitutions and deletions located on α domain, and 4 CHB in RCH patients carried amino acid substitutions and deletions located in β –domain, 72.7% vs. 36.3%, respectively. There was not a statistically significant difference between RCH patients with and without CHB (P = 0.473).