Clinical Scoring System for Silver-Russell Syndrome
Clinical Scoring System for Silver-Russell Syndrome
The 69 study participants were examined by two paediatric endocrinologists with considerable experience in the diagnosis and care of patients with SRS. These physicians together agreed upon a series of phenotypical characteristics for each patient. The presence or absence of a protruding forehead was determined during the initial examination. For patients over the age of 3 years, the two physicians also evaluated this facial characteristic together, on photos of the child aged 1–3 years (face and profile). This method made it possible to collect homogeneous phenotypical data confirmed by two examiners. Following the review of the photos, each patient was then scored for the new NH-CSS (see online supplementary table S1 http://jmg.bmj.com/content/52/7/446/suppl/DC1). Sixty of the 69 patients (35 boys and 25 girls; mean age 6.85 years) included in the study satisfied at least four of the criteria (mean factor frequency=5.29) of the NH-CSS and were consequently classified as 'Likely-SRS'. Nine patients (two boys and seven girls; mean age 4.96 years) were considered as satisfying up to three of these criteria (mean factor frequency=2.89) and were classified as 'Unlikely-SRS'.
We then compared these two groups (Likely-SRS and Unlikely-SRS). We initially studied only those patients for whom we had data for all six criteria (n=61). We compared 52 patients from the Likely-SRS group with the 9 patients from the Unlikely-SRS group. As expected, these two groups displayed statistically significant clinical differences (t test; p=0.000). We also compared the two groups for each NH-CSS criteria separately. The two groups did not differ significantly in terms of birth weight or length or postnatal growth failure. By contrast, they differed significantly for the other four criteria of the NH-CSS (Pearson's χ; p=0.000) ( Table 1 ).
11p15 Methylation and mUPD7 Screening. We assessed 11p15 ICR1 region methylation status and mUPD7 in the 69 patients. We detected 11p15 ICR1 and mUPD7 abnormalities by carrying out ASMM RTQ-PCR to determine the methylation status of ICR1 CBS2 and H19DMR (H19 promoter) for 11p15 ICR1 and of PEG/MEST in the 7p32 region for mUPD7. We identified molecular abnormalities in 46 of the 60 (76.7%) Likely-SRS patients (35 (58.3%) (22 boys and 13 girls; mean age 6.9 years) due to 11p15 ICR1 hypomethylation and 11 (18.3%) (7 boys and 5 girls; mean age 7.69 years) due to mUPD7). Fourteen of the 60 Likely-SRS patients (23.3%) (6 boys and 8 girls; mean age 5.79 years) were negative for both abnormalities and are described hereafter as Likely-SRS double-negatives (L-SRS-dblneg). No molecular abnormality of either the 11p15 ICR1 region or mUPD7 was identified in eight of the nine (88.9%) patients in the Unlikely-SRS group (UL-SRS-dblneg). However, one patient who was positive for three criteria of the NH-CSS was found to have mUPD7.
Overall, NH-CSS successfully picked up 97.9% of the patients with known molecular abnormalities associated with SRS (100% of the 35 11p15 ICR1 patients and 91.7% of the mUPD7 patients), missing only a single mUPD7 patient.
Multilocus Imprinting Disturbance in Patients With SRS With ICR1 11p15 Hypomethylation. Multilocus imprinting disturbance (MLID) is increasingly being reported in human imprinting diseases, including SRS. We therefore screened for MLID in this well clinically characterised cohort of patients with SRS. We investigated the methylation status of 11p15 ICR2, the DLK1/GTL2 IG-DMR locus on chromosome 14q32, ZAC1 DMR on 6q24, SNRPN on 15q11.2 and the GNAS locus (XLa DMR, AB DMR and NESP55 DMR) on 20q13.32, by ASMM RTQ-PCR. Four of 35 patients (11.4%) were found to display MLID: one with hypomethylation at ZAC1 DMR (MI=36%; range of normal values 45–57%); one with a methylation profile similar to that found in hypoparathyroidism type 1 b (PHP1b) patients (ie, hypomethylation at XLa DMR (MI=18%) and AB DMR (MI=21%) and hypermethylation at NESP55 DMR (MI=73%)) and two patients displayed hypomethylation of the DLK1/GTL2 IG-DMR locus (MIs=17% and 8%).
Further Findings. For all patients negative for the conventional molecular aetiologies of SRS, we searched for 11p15 ICR2 duplication, CDKN1C mutation or copy number variation (CNV) or imprinting abnormalities in other imprinted regions that might account for the SRS phenotype in the L-SRS-dblneg group or the growth retardation observed in the UL-SRS-dblneg group. We carried out genome-wide screening of DNA with the CytoSNP12 microarray from all the patients of the L-SRS-dblneg and UL-SRS-dblneg groups. Methylation abnormalities in imprinted regions were assessed by ASMM RTQ-PCR. We identified four independent molecular defects in four patients from the L-SRS-dblneg group. Two patients displayed maternal UPD, one of chromosome 16 (mUPD16) and the other of chromosome 20 (mUPD20). A third patient carried a de novo 1q21 3 Mb microdeletion (figure 2). The fourth patient displayed a loss of methylation at the DLK1/GTL2 IG-DMR locus; and the CytoSNP12 microarray excluded mUPD14, microdeletion or microduplication involving the 14q32 domain.
(Enlarge Image)
Figure 2.
Chromosomal abnormalities found in Likely-Silver-Russell syndrome (SRS) double-negative patients (left panel) and Unlikely-SRS double-negative patients (right panel).
In the UL-SRS-dblneg group, we identified three independent chromosomal rearrangements in three patients: one patient carried a de novo 20.2 Mb 1q24.3q31.2 microdeletion, the second carried a de novo 5.2 Mb 11p13-p12 microdeletion centromeric to the WT1 gene and the third carried a paternally transmitted 2.6 Mb 22q11.21 microduplication.
The clinical features of these four L-SRS-dblneg and three UL-SRS-dblneg patients plus the UL-SRS mUPD7 patient are summarised in online supplementary table S7 and supplementary data http://jmg.bmj.com/content/52/7/446/suppl/DC1.
We reclassified the Likely-SRS and Unlikely-SRS patients according to the molecular data which results in four groups (11p15 hypomethylation, mUPD7, L-SRS-dblneg and Unlikely-SRS) and then investigated the differences between them in terms of NH-CSS factors. We excluded the mUPD7 false-negative patient from this analysis for the sake of clarity. However, it should be noted that its exclusion had no effect on the results of the statistical analysis (data not shown). Overall scores differed significantly between the four groups ( Table 2 ; p=0.000). By including only subjects for whom data were available for all six factors, we ensured that comparisons between groups in terms of the overall mean number of NH-CSS positive criteria were carried out correctly. We then compared scoring system factors separately across the four groups. Statistically significant differences between the four groups were found for all the criteria except for one of these factors, postnatal growth failure (χ test; p=0.06).
Pairwise comparison analyses showed that the 11p15 ICR1 hypomethylated group was statistically different overall and for all six factors considered separately. The Unlikely-SRS group also differed clinically from the other groups (see online supplementary table S2 http://jmg.bmj.com/content/52/7/446/suppl/DC1). Relative macrocephaly was present in a significantly higher percentage of individuals from the SRS 11p15 and SRS mUPD7 groups than in the UL-SRS group (see online supplementary table S3 http://jmg.bmj.com/content/52/7/446/suppl/DC1). The percentage of subjects with a protruding forehead was also significantly higher in the SRS 11p15 and SRS mUPD7 groups than in the UL-SRS group (see online supplementary table S4 http://jmg.bmj.com/content/52/7/446/suppl/DC1). The percentage of subjects with body asymmetry was also significantly higher in the SRS 11p15 group than in all the other groups but this percentage was significantly higher in the L-SRS-dblneg than in the UL-SRS group (see online supplementary table S5 http://jmg.bmj.com/content/52/7/446/suppl/DC1). Finally, feeding difficulties and low BMI were significantly more frequent in the SRS 11p15, SRS mUPD7 and L-SRS-dblneg groups than in the UL-SRS group (see online supplementary table S6 http://jmg.bmj.com/content/52/7/446/suppl/DC1).
The NH-CSS failed to identify only one patient for whom subsequent molecular analysis demonstrated SRS mUPD7. Therefore, we sought to identify additional characteristics useful for the clinical diagnosis of SRS for borderline cases with the NH-CSS score of 3–4. We compared the four molecular groups for a number of cognitive and physical variables previously reported or considered by the investigators (IN and MDH) to be common in children with SRS (see online supplementary table S8 http://jmg.bmj.com/content/52/7/446/suppl/DC1). The four groups differed significantly in terms of a number of quantifiable physical characteristics; however, incidence calculations showed that the significance of these characteristics was often limited to specific groups (see online supplementary data http://jmg.bmj.com/content/52/7/446/suppl/DC1).
We compared our NH-CSS with the scoring system previously reported by Netchine et al and the Birmingham clinical scoring system. We applied these three clinical scoring systems to the 69 patients studied here. We considered only the two common molecular aetiologies of SRS, mUPD7 and 11p15 ICR1 hypomethylation, for SRS-positive molecular testing. Our system classified 60 patients as Likely-SRS ( Table 3 ), whereas the Netchine et al and Birmingham systems classified only 55 and 47 patients, respectively, as Likely-SRS. The number of Unlikely-SRS patients was therefore higher with these two systems, 14 and 16 patients, respectively than with our new system, 9 patients.
Interestingly, whereas our new scoring system and that of Netchine et al identified 100% of the patients with 11p15 ICR1 hypomethylation, the Birmingham scoring system missed two of the patients (93.7% of identification). In addition, our new scoring system identified all but one of the mUPD7 patients (91.7% identified), whereas the Netchine et al system missed four mUPD7 (66.7% identified) and the Birmingham system missed five mUPD7 (54.6% identified). Overall, the number of false-negative results was reduced in the new scoring system. The Birmingham system uses only four factors making it more prone to the negative effect of missing data points. This resulted in the exclusion of six additional patients due to missing data including three 11p15 ICR1 hypomethylation and one mUPD7 patient. Overall, the Birmingham system failed to identify 11 subjects with molecularly confirmed SRS from our study population.
The new NH-CSS was more sensitive (97.9%) than the Netchine et al (91.5%) and Birmingham (83.7%) clinical scoring systems ( Table 4 ). By contrast, our new NH-CSS is less specific (specificity=36%) than the other systems. The three tests had similar positive predictive values, but the NH-CSS had the highest negative predictive value (NPV, 88.9%). Thus, we can have a high degree of confidence that a score of less than four on the NH-CSS screen has a high likelihood of being truely SRS-negative. The Birmingham scoring system had the lowest NPV of the three clinical scoring systems tested (56.3%), due to the number of patients with positive molecular findings who would not have been tested if the decision to test were based on this scoring system.
Results
Netchine-Harbison Clinical Scoring System Results
The 69 study participants were examined by two paediatric endocrinologists with considerable experience in the diagnosis and care of patients with SRS. These physicians together agreed upon a series of phenotypical characteristics for each patient. The presence or absence of a protruding forehead was determined during the initial examination. For patients over the age of 3 years, the two physicians also evaluated this facial characteristic together, on photos of the child aged 1–3 years (face and profile). This method made it possible to collect homogeneous phenotypical data confirmed by two examiners. Following the review of the photos, each patient was then scored for the new NH-CSS (see online supplementary table S1 http://jmg.bmj.com/content/52/7/446/suppl/DC1). Sixty of the 69 patients (35 boys and 25 girls; mean age 6.85 years) included in the study satisfied at least four of the criteria (mean factor frequency=5.29) of the NH-CSS and were consequently classified as 'Likely-SRS'. Nine patients (two boys and seven girls; mean age 4.96 years) were considered as satisfying up to three of these criteria (mean factor frequency=2.89) and were classified as 'Unlikely-SRS'.
We then compared these two groups (Likely-SRS and Unlikely-SRS). We initially studied only those patients for whom we had data for all six criteria (n=61). We compared 52 patients from the Likely-SRS group with the 9 patients from the Unlikely-SRS group. As expected, these two groups displayed statistically significant clinical differences (t test; p=0.000). We also compared the two groups for each NH-CSS criteria separately. The two groups did not differ significantly in terms of birth weight or length or postnatal growth failure. By contrast, they differed significantly for the other four criteria of the NH-CSS (Pearson's χ; p=0.000) ( Table 1 ).
Molecular Analysis
11p15 Methylation and mUPD7 Screening. We assessed 11p15 ICR1 region methylation status and mUPD7 in the 69 patients. We detected 11p15 ICR1 and mUPD7 abnormalities by carrying out ASMM RTQ-PCR to determine the methylation status of ICR1 CBS2 and H19DMR (H19 promoter) for 11p15 ICR1 and of PEG/MEST in the 7p32 region for mUPD7. We identified molecular abnormalities in 46 of the 60 (76.7%) Likely-SRS patients (35 (58.3%) (22 boys and 13 girls; mean age 6.9 years) due to 11p15 ICR1 hypomethylation and 11 (18.3%) (7 boys and 5 girls; mean age 7.69 years) due to mUPD7). Fourteen of the 60 Likely-SRS patients (23.3%) (6 boys and 8 girls; mean age 5.79 years) were negative for both abnormalities and are described hereafter as Likely-SRS double-negatives (L-SRS-dblneg). No molecular abnormality of either the 11p15 ICR1 region or mUPD7 was identified in eight of the nine (88.9%) patients in the Unlikely-SRS group (UL-SRS-dblneg). However, one patient who was positive for three criteria of the NH-CSS was found to have mUPD7.
Overall, NH-CSS successfully picked up 97.9% of the patients with known molecular abnormalities associated with SRS (100% of the 35 11p15 ICR1 patients and 91.7% of the mUPD7 patients), missing only a single mUPD7 patient.
Multilocus Imprinting Disturbance in Patients With SRS With ICR1 11p15 Hypomethylation. Multilocus imprinting disturbance (MLID) is increasingly being reported in human imprinting diseases, including SRS. We therefore screened for MLID in this well clinically characterised cohort of patients with SRS. We investigated the methylation status of 11p15 ICR2, the DLK1/GTL2 IG-DMR locus on chromosome 14q32, ZAC1 DMR on 6q24, SNRPN on 15q11.2 and the GNAS locus (XLa DMR, AB DMR and NESP55 DMR) on 20q13.32, by ASMM RTQ-PCR. Four of 35 patients (11.4%) were found to display MLID: one with hypomethylation at ZAC1 DMR (MI=36%; range of normal values 45–57%); one with a methylation profile similar to that found in hypoparathyroidism type 1 b (PHP1b) patients (ie, hypomethylation at XLa DMR (MI=18%) and AB DMR (MI=21%) and hypermethylation at NESP55 DMR (MI=73%)) and two patients displayed hypomethylation of the DLK1/GTL2 IG-DMR locus (MIs=17% and 8%).
Further Findings. For all patients negative for the conventional molecular aetiologies of SRS, we searched for 11p15 ICR2 duplication, CDKN1C mutation or copy number variation (CNV) or imprinting abnormalities in other imprinted regions that might account for the SRS phenotype in the L-SRS-dblneg group or the growth retardation observed in the UL-SRS-dblneg group. We carried out genome-wide screening of DNA with the CytoSNP12 microarray from all the patients of the L-SRS-dblneg and UL-SRS-dblneg groups. Methylation abnormalities in imprinted regions were assessed by ASMM RTQ-PCR. We identified four independent molecular defects in four patients from the L-SRS-dblneg group. Two patients displayed maternal UPD, one of chromosome 16 (mUPD16) and the other of chromosome 20 (mUPD20). A third patient carried a de novo 1q21 3 Mb microdeletion (figure 2). The fourth patient displayed a loss of methylation at the DLK1/GTL2 IG-DMR locus; and the CytoSNP12 microarray excluded mUPD14, microdeletion or microduplication involving the 14q32 domain.
(Enlarge Image)
Figure 2.
Chromosomal abnormalities found in Likely-Silver-Russell syndrome (SRS) double-negative patients (left panel) and Unlikely-SRS double-negative patients (right panel).
In the UL-SRS-dblneg group, we identified three independent chromosomal rearrangements in three patients: one patient carried a de novo 20.2 Mb 1q24.3q31.2 microdeletion, the second carried a de novo 5.2 Mb 11p13-p12 microdeletion centromeric to the WT1 gene and the third carried a paternally transmitted 2.6 Mb 22q11.21 microduplication.
The clinical features of these four L-SRS-dblneg and three UL-SRS-dblneg patients plus the UL-SRS mUPD7 patient are summarised in online supplementary table S7 and supplementary data http://jmg.bmj.com/content/52/7/446/suppl/DC1.
Comparison of NH-CSS Criteria Between the Different Molecular Groups
We reclassified the Likely-SRS and Unlikely-SRS patients according to the molecular data which results in four groups (11p15 hypomethylation, mUPD7, L-SRS-dblneg and Unlikely-SRS) and then investigated the differences between them in terms of NH-CSS factors. We excluded the mUPD7 false-negative patient from this analysis for the sake of clarity. However, it should be noted that its exclusion had no effect on the results of the statistical analysis (data not shown). Overall scores differed significantly between the four groups ( Table 2 ; p=0.000). By including only subjects for whom data were available for all six factors, we ensured that comparisons between groups in terms of the overall mean number of NH-CSS positive criteria were carried out correctly. We then compared scoring system factors separately across the four groups. Statistically significant differences between the four groups were found for all the criteria except for one of these factors, postnatal growth failure (χ test; p=0.06).
Pairwise comparison analyses showed that the 11p15 ICR1 hypomethylated group was statistically different overall and for all six factors considered separately. The Unlikely-SRS group also differed clinically from the other groups (see online supplementary table S2 http://jmg.bmj.com/content/52/7/446/suppl/DC1). Relative macrocephaly was present in a significantly higher percentage of individuals from the SRS 11p15 and SRS mUPD7 groups than in the UL-SRS group (see online supplementary table S3 http://jmg.bmj.com/content/52/7/446/suppl/DC1). The percentage of subjects with a protruding forehead was also significantly higher in the SRS 11p15 and SRS mUPD7 groups than in the UL-SRS group (see online supplementary table S4 http://jmg.bmj.com/content/52/7/446/suppl/DC1). The percentage of subjects with body asymmetry was also significantly higher in the SRS 11p15 group than in all the other groups but this percentage was significantly higher in the L-SRS-dblneg than in the UL-SRS group (see online supplementary table S5 http://jmg.bmj.com/content/52/7/446/suppl/DC1). Finally, feeding difficulties and low BMI were significantly more frequent in the SRS 11p15, SRS mUPD7 and L-SRS-dblneg groups than in the UL-SRS group (see online supplementary table S6 http://jmg.bmj.com/content/52/7/446/suppl/DC1).
The NH-CSS failed to identify only one patient for whom subsequent molecular analysis demonstrated SRS mUPD7. Therefore, we sought to identify additional characteristics useful for the clinical diagnosis of SRS for borderline cases with the NH-CSS score of 3–4. We compared the four molecular groups for a number of cognitive and physical variables previously reported or considered by the investigators (IN and MDH) to be common in children with SRS (see online supplementary table S8 http://jmg.bmj.com/content/52/7/446/suppl/DC1). The four groups differed significantly in terms of a number of quantifiable physical characteristics; however, incidence calculations showed that the significance of these characteristics was often limited to specific groups (see online supplementary data http://jmg.bmj.com/content/52/7/446/suppl/DC1).
Comparison of the Clinical Scoring Systems for Their Sensitivity and Specificity
We compared our NH-CSS with the scoring system previously reported by Netchine et al and the Birmingham clinical scoring system. We applied these three clinical scoring systems to the 69 patients studied here. We considered only the two common molecular aetiologies of SRS, mUPD7 and 11p15 ICR1 hypomethylation, for SRS-positive molecular testing. Our system classified 60 patients as Likely-SRS ( Table 3 ), whereas the Netchine et al and Birmingham systems classified only 55 and 47 patients, respectively, as Likely-SRS. The number of Unlikely-SRS patients was therefore higher with these two systems, 14 and 16 patients, respectively than with our new system, 9 patients.
Interestingly, whereas our new scoring system and that of Netchine et al identified 100% of the patients with 11p15 ICR1 hypomethylation, the Birmingham scoring system missed two of the patients (93.7% of identification). In addition, our new scoring system identified all but one of the mUPD7 patients (91.7% identified), whereas the Netchine et al system missed four mUPD7 (66.7% identified) and the Birmingham system missed five mUPD7 (54.6% identified). Overall, the number of false-negative results was reduced in the new scoring system. The Birmingham system uses only four factors making it more prone to the negative effect of missing data points. This resulted in the exclusion of six additional patients due to missing data including three 11p15 ICR1 hypomethylation and one mUPD7 patient. Overall, the Birmingham system failed to identify 11 subjects with molecularly confirmed SRS from our study population.
The new NH-CSS was more sensitive (97.9%) than the Netchine et al (91.5%) and Birmingham (83.7%) clinical scoring systems ( Table 4 ). By contrast, our new NH-CSS is less specific (specificity=36%) than the other systems. The three tests had similar positive predictive values, but the NH-CSS had the highest negative predictive value (NPV, 88.9%). Thus, we can have a high degree of confidence that a score of less than four on the NH-CSS screen has a high likelihood of being truely SRS-negative. The Birmingham scoring system had the lowest NPV of the three clinical scoring systems tested (56.3%), due to the number of patients with positive molecular findings who would not have been tested if the decision to test were based on this scoring system.
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