Contribution of gene mutations to Silver-Russell syndrome phenotype: multigene sequencing analysis in 92 etiology-unknown patients

Abstract

Background: Silver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features. Major (epi)genetic causes of SRS are loss of methylation on chromosome 11p15 (11p15 LOM) and maternal uniparental disomy of chromosome 7 (upd(7)mat). However, IGF2, CDKN1C, HMGA2, and PLAG1 mutations infrequently cause SRS. In addition, other imprinting disturbances, pathogenic copy number variations (PCNVs), and monogenic disorders sometimes lead to SRS phenotype. This study aimed to clarify the frequency and clinical features of the patients with gene mutations among etiology-unknown patients with SRS phenotype.

Results: Multigene sequencing was performed in 92 out of 336 patients referred to us for genetic testing for SRS. The clinical features of the patients were evaluated based on the Netchine-Harbison clinical scoring system. None of the patients showed 11p15 LOM, upd(7)mat, abnormal methylation levels for six differentially methylated regions (DMRs), namely, PLAGL1:alt-TSS-DMR on chromosome 6, KCNQ1OT1:TSS-DMR on chromosome 11, MEG3/DLK1:IG-DMR on chromosome 14, MEG3:TSS-DMR on chromosome 14, SNURF:TSS-DMR on chromosome 15, and GNAS A/B:TSS-DMR on chromosome 20, PCNVs, or maternal uniparental disomy of chromosome 16. Using next-generation sequencing and Sanger sequencing, we screened four SRS-causative genes and 406 genes related to growth failure and/or skeletal dysplasia. We identified four pathogenic or likely pathogenic variants in responsible genes for SRS (4.3%: IGF2 in two patients, CDKN1C, and PLAG1), and five pathogenic variants in causative genes for known genetic syndromes presenting with growth failure (5.4%: IGF1R abnormality (IGF1R), SHORT syndrome (PIK3R1), Floating-Harbor syndrome (SRCAP), Pitt-Hopkins syndrome (TCF4), and Noonan syndrome (PTPN11)). Functional analysis indicated the pathogenicity of the CDKN1C variant. The variants we detected in CDKN1C and PLAG1 were the second and third variants leading to SRS, respectively. Our patients with CDKN1C and PLAG1 variants showed similar phenotypes to previously reported patients. Furthermore, our data confirmed IGF1R abnormality, SHORT syndrome, and Floating-Harbor syndrome are differential diagnoses of SRS because of the shared phenotypes among these syndromes and SRS. On the other hand, the patients with pathogenic variants in causative genes for Pitt-Hopkins syndrome and Noonan syndrome were atypical of these syndromes and showed partial clinical features of SRS.

Conclusions: We identified nine patients (9.8%) with pathogenic or likely pathogenic variants out of 92 etiology-unknown patients with SRS phenotype. This study expands the molecular spectrum of SRS phenotype.

Keywords: CDKN1C; Floating-Harbor syndrome; Functional analysis; IGF1R; Multigene sequencing; Noonan syndrome; PLAG1; Pitt-Hopkins syndrome; SHORT syndrome; Silver-Russell syndrome.

Fig. 1

Flowchart of inclusion criteria. A total of 336 patients were referred to us for genetic testing for Silver-Russell syndrome (SRS) from 2002 to 2018. Our study included 92 patients without pathogenic copy number variations or abnormal methylation levels for ten differentially methylated regions (DMRs), namely, H19/IGF2:IG-DMR, PEG10:TSS-DMR, MEST:alt-TSS-DMR, PLAGL1:alt-TSS-DMR, KCNQ1OT1:TSS-DMR, MEG3/DLK1:IG-DMR, MEG3:TSS-DMR, SNURF:TSS-DMR, ZNF597:TSS-DMR, and GNAS A/B:TSS-DMR. 11p15 LOM, loss of methylation on chromosome 11p15; upd(7)mat, maternal uniparental disomy of chromosome 7; NH-CSS, Netchine-Harbison clinical scoring system; Chr, chromosome; upd(20)mat, maternal uniparental disomy of chromosome 20; upd(6)mat, maternal uniparental disomy of chromosome 6; upd(11)mat, maternal uniparental disomy of chromosome 11; upd(16)mat, maternal uniparental disomy of chromosome 16. *We evaluated clinical features of only a part of the patients according to the Netchine-Harbison clinical scoring system. **The duplicated region of two patients with 11p15 duplications did not include the H19/IGF2:IG-DMR. Thus, these patients showed normal methylation levels of the H19/IGF2:IG-DMR. The duplicated region of the remaining one patient included the H19/IGF2:IG-DMR. The methylation level of the H19/IGF2:IG-DMR in this patient was low normal, and we did not recognize 11p15 LOM. ***We began upd(16)mat screening in 2016. As such, we performed upd(16)mat screening for only a part of the patients with pathogenic copy number variations and patients with abnormal methylation levels of the DMRs related to known imprinting disorders before 2016

Fig. 2

Clinical findings of the patients identified in this study. a Pedigrees of patients with variants inherited from their parents. b Growth charts. c Photographs of the patients and the mother of patient 4. Patients 1 and 2 were already reported [12]. SDS, standard deviation score; GH, growth hormone

Fig. 3

Results of Western blot analysis. The doxycycline-inducible protein expression level of Arg316Gln-CDKN1C was higher than that of WT-CDKN1C. The experiment was conducted in triplicate. WT, wildtype