3.1. Patients' Selection and Initial Assessment
Six RTT patients between four and eleven years old with the classical form of RTT were treated for this study (see ). The subjects studied are the following: S1: 4.8 years, MeCP2 mutation: deletion of exons 3 and 4, with a severity of illness of 4 (CGI); S2: 8.10 years, MeCP2 mutation: T158M (missense), severity of illness of 6 (CGI); S3: 5.8 years, MeCP2 mutation: R133C (missense) severity of illness of 3 (CGI); S4: 5.9 years, MeCP2 mutation: C13insGCCGC in exon 1 (frameshift), severity of illness of 3 (CGI); S5: 4.7, MeCP2 mutation: R270X (nonsense), severity of illness of 5 (CGI); S6: 10.8, MeCP2 mutation 1055 del 12 + 1157 del 44 (frameshift), severity of illness of 3 (CGI). For a summary of subjects and mutations see . At the start of IGF1 administration, S1, S2, and S3 were already under treatment for epilepsy with valproic acid at the dosage of 20
3.2. Blood Parameters
During hospitalizations the blood parameters were measured. In particular, blood count (leucocytes, haematocrit, haemoglobin, neutrophils, and lymphocytes) revealed no significant changes during the treatment while serum levels of IGF1 increased—as expected—during the treatment and went back to baseline values after the end of IGF1 administration (Wilcoxon test, P value = 0.03). The initial endogenous levels of IGF1 were within the age range for each patient (). Glucose and growth hormone levels stayed within the reference values in each patient, while insulin level dropped below the expected level in S1 and S2 during IGF1 administration.
Figure 1 IGF1 serum levels during the treatment. IGF1 levels in the blood were tested at each hospitalization: D0 (first day of treatment, before IGF administration), D90 (90 days after the first IGF1 administration), D180 (180 days after the first IGF administration, (more ...)
Blood gas values at baseline were abnormal prior to commencing treatment, consistent with the respiratory pathology of RTT, where patients present irregular episodes of hyperventilation, breath holding, and apneas. Changes in venous blood gas parameters (pO2, pCO2, pH, O2Hb, HHb, CHCO3, and SatO2) revealed changes in all patients during IGF1 treatment, but no definite pattern is visible and statistical analysis reveals no significant differences between sessions. The specific values are reported in : pO2 was initially normal for S1, but it reached higher values after IGF administration and returned within the range after the suspension of the drug. Conversely, S3, S4, and S5 had always regular values for pO2, and so it was for S2, with the exception of D270. For S6 the recorded values were always above the range. pCO2 measurements were always normal for S1, with the exception of D90, when they went below the limits. S2 had an abnormal pCO2 values before starting the treatment, they went in the normal range during the treatment and returned below the limits after the suspension of the drug. S3 had normal pCO2 values below the limits at the beginning of the treatment but the values were in the normal range in subsequent sessions. S4 had normal pCO2 values at D0 and D90 and the values went above the range after D180. For S5 the pCO2 value was always above the limits while it was constantly in the normal range for S6. The pH values were in the normal range in the majority of cases with the exception of S2 at 270, S4 at D180 and D270, and S5 at D90. The measurements of O2 Hb and Hbb were normal for all the patients across all the sessions and therefore they are not shown in . Conversely, the CHCO3 values were altered in the majority of cases with the exception of S6, who had always normal values. The SatO2 initially was abnormally high for S1 and returned to normal values after D270, while for S2 the measurements were always normal with the exception of D90, when it became abnormally low. S3 and S4 had initial values above the range, but they went back to normal in the following sessions. For S5 and S6 the SatO2 values were constantly abnormal (below the range for S5 and above the range for S6).
Gas parameters. This table reports the measurements of gas parameters for each patient at D0 and following hospitalization. NR: not registered. NA: not available.
3.3. Growth Parameters
We measured the growth parameters: body weight, height, and head circumference (). In all of the patients we observed an increase in body weight, with the exception of S2, who had a reduction in the body weight following 3 months of treatment. Across all patients, there was a significant increase in body weight between D0 and D90 (Wilcoxon test, P value = 0.03) with the exception of S2, who was overweight at the beginning of the treatment, and she reduced body weight. The growth trend continued even after the treatment was suspended, and this is true also for S2 who continued to lose weight, although she remained overweight despite the IGF1-driven weight loss.
Figure 2 Growth parameters progression during IGF1 treatment. The figure reports the increase/decrease in weights (a), height (b), and head circumference (c) in each patient for one year, starting from Day0, D0 (first day of treatment, before IGF administration). (more ...)
Patients showed a significant increase in height between D0 and D90 (Wilcoxon test, P value = 0.03) that continued even after the end of the treatment. Head circumference showed a trend of increase (though not significant) in all the patients with the exception of S5, who was unchanged ().
It is important to stress that even if there was an increase in the parameters examined, the growth remains impaired to the development of normal age-matched girls.
3.4. Glycemia Measurements
Each family received a kit for the detection of blood level of glucose, and the glucose concentration in the blood was measured by the caretakers every day for the after 60 minutes from the IGF1 administration, in addition, hypoglycaemia was evaluated as part of blood tests during periodic hospitalization. After few weeks, the glycaemia was measured only if one of the symptoms was observed (dizziness, sweating, headache, fatigue, nausea, irritability, difficulty concentrating, and tachycardia).
Hypoglycaemia was cautiously defined as blood levels of glucose below 50
mg random. None of the patients went below the set threshold; however, one patient (S1) was reported to have a glucose value of 55 at 118 days from the beginning of the treatment (). The measurement followed a period of vomiting and fasting. Treatment was suspended and restarted after 24
hours, following several normal measurements of glucose level.
Figure 3 Glycemia values in Subject 1. Glucose blood levels were registered by the caregiver and recorded in the journal. The measurements were taken with a home device that the caregiver was trained to use on D0. On the X-axis are reported the days when the single (more ...)
3.5. Seizures Outcome during IGF Treatment
Some of the girls (S1, S2, and S3) in this study had seizures before starting the IGF treatment, and they were regularly treated with valproate. During crisis, S1 had staring eyes and muscular rigidity; S2's crisis started with cyanosis, followed by lateral limbs hypertonicity, pallor, loss of consciousness, and hypotonia. S3 had shaking, hypotonia followed by muscular rigidity, staring eyes, guttural sounds, and sleep. IGF1 treatment was administered alongside regular antiepileptic medication (AED) as described without changing the therapy for seizures. Seizure frequency and AED therapy remained unchanged, suggesting no interaction with valproate. S5, S4, and S6 did not report seizures before the IGF1 treatment. One of them, S6, never showed seizures, while S4 and S5 showed seizures during the IGF treatment. S4 had a single episode of seizures after three months from the start of the treatment, characterized by jaw clonic jerks and guttural vocalization for less than one minute, and AED was not indicated. Following discontinuation of IGF treatment, S4 had repeated and prolonged episodes of the described seizures and valproate was commenced.
S5 developed seizures two months after the start of IGF1 therapy and was commenced on valproate ().
Figure 4 Seizures outcome during IGF1 treatment. The figure shows the outcome of seizures during one year starting with D0. The dotted lines represent the start (D0) and the end (D180) of the treatment. Each patient is color-coded: S1: blue, S2: red, S3: green, (more ...)
3.6. Cardiorespiratory Activity
Patients with classic RTT have impaired cardiorespiratory function. Four out of six patients presented with superficial breathing and apneas (S4, S2, and S6). One patient, S3, presented with “enhanced phenotype,” that is, tachypnea and hyperpnea. S1 had an alternating pathology of apneas and tachypneas with no particular prevalence of one. In addition, three of the six patients, S4, S2, and S3, had a high prevalence of Valsalva manoluvre.
Evaluation of cardiac function included measuring R-R distance on ECG, heart rate, and vagal tone. No significant changes in these measurements were detected during IGF1 treatment.
To evaluate the breathing activity, during the hospital visits, we monitored the respiration for one hour, we performed a clinical evaluation of the respiratory activity, and we measured the following parameters: breath holding, apnea, Valsalva's manoeuvre, and normal respiration. The measurements were used for the clinical evaluation of the breathing activity, which is represented in . The effects on the single parameters and blood gas analysis (pO2, pCO2, and pH) were highly dependent on the patient and did not show any significant trend (). Clinical assessment, after evaluation of the general respiratory pattern, suggested an improvement during IGF1 treatment with a regression after the end of the treatment (). We compared the clinical assessments scores for each patient between consecutive sessions and we found a significant improvement between D0 and D90 (Wilcoxon test, P value = 0.04).
Figure 5 Clinical assessment of IGF1 treatment on breathing. The graph reports the clinical evaluation of respiratory activity at each visit compared with the previous observation. D0 (first day of treatment, before IGF administration), D90 (90 days after the (more ...)
3.7. Bone Density
We then measured the effect of IGF1 treatment on bone structure [31
]. Bone density was measured at baseline and after one year from the beginning of the treatment (D365). The readings for S5 are not available for technical reasons. S3 and S6 had increased bone density following treatment. Bone density was unchanged in the remaining subjects ().
Figure 6 Effects of IGF1 treatment on bone density. (a): row values for bone density for each patient at D0 (empty histogram) and D365 (filled histogram). D0 (first day of treatment, before IGF administration), D90 (90 days after the first IGF1 administration), (more ...)
3.8. Evaluation of Brain Activity—EEG
Patients were assessed for brain activity on D0 and after one year (D365). Ten electrodes were placed on the scalp and the following signals recorded: Fp2-C, C4-T4, T4-O2, O2-C4, Fp1-C3, C3-T3, T3-O1, and O1-C3. The treated patients were compared to a group of six untreated RTT patients (age matched and with comparable RTT clinical stage). We considered the signal of each derivation, and we evaluated both the amplitude and the spectrum of frequency for each derivation (). We focused on delta and theta signals as they are more relevant for RTT [32
]. The analysis of amplitude revealed that the mean delta amplitude was not different between treated and not treated patients at the beginning and at the end of the treatment and that IGF1 did not cause any change in this parameter. The frequence of delta waves remained unvaried for both treated and untreated patients between the beginning and the end of the treatment.
Figure 7 Effects of IGF1 treatment on brain activity. (a) Spectrum of frequencies (left) and EEG trace (right) for the site C4-T4 in all the treated subjects S1–S6 at D0 and D365. (b) Spectrum of frequencies (left) and EEG trace (right) for the site C4-T4 (more ...)
The amplitude of teta waves was not significantly different between D0 and D365 (). The frequence of theta waves was significantly higher in treated versus untreated patients at D365 for the electrode C4-T4 (Wilcoxon test, P value = 0.04) ().
3.9. Clinical Evaluation according to International Severity Scale
During hospital evaluation the patients were scored for the parameters of the International Severity Scale (ISS). This scale takes into account parameters of growth and development (growth, body weight, and height), locomotor apparatus (muscle tone and spinal column), locomotor ability (deambulation, stereotypes, and purposeful use of hands), cortical functions (cognition, epilepsy, and speech), and autonomic functions (respiration, circulation, digestive system, and sleep). The lower the score on this scale, the better the abilities. At the end of IGF treatment (D180) we observed a reduction (improvement) in the ISS score for the patients S6, S4, S3, and S2 (), while for S5 and S1 we observed no changes or even a slight worsening. After six months from the end of IGF1 treatment (D365) there was an improvement in the ISS score for S4 and S6, while for S3 the ISS score was the same as that at D0 and for S1, S2, and S5 was higher (). The improvement in ISS parameter was not significant considering the whole cohort of treated subjects.
Figure 8 Effects of IGF1 treatment on International Severity Scale (ISS). The graph reports the clinical evaluation of the parameters of the International Severity Scale (ISS). D0 (first day of treatment, before IGF administration), D90 (90 days after the first (more ...)
3.10. Sleep, Nutrition, Language, and Cognition
At the first visit the families were given a journal for filling in information regarding sleep, nutrition, abnormal behaviour. According to the annotations non particular changes occurred during or after IGF1 treatment in the sleep and eating behaviour of the patients.
No changes were observed in the language abilities although parents and caregivers report an increase in the communication abilities, that is, the girls were more able to communicate their intentions/needs. These improvements are in line with a more general improvement in cognitive abilities and attention that has been reported for all the patients even after the end of the treatment. Both teachers and family members report that the patients were more able to engage with the environment and to interact with friends/family members. However, an appropriate test is required for assessing the cognitive benefits of IGF1. Such measurements are beyond the purpose of this study.
3.11. Side Effects and Parents Assessments
No major side effects were observed in any patient; however, we report mammary hyperplasia for S1, first on the right side, after 40 days from the beginning of the IGF treatment, then on the left side (). Ultrasound analysis did not reveal additional morbidity. The hyperplasia regressed on both sides within two weeks without changes to the IGF treatment.
Figure 9 IGF1 treatment is safe and tolerated by patients. Representation of the possible side effects of IGF1 in all of the treated patients: hypoglycemia (H-G), tonsillar hypertrophy (H-T-T), mammary hyperplasia (H-M), and seizures (S) at different times after (more ...)
We asked the parents after one year if they would repeat the IGF treatment. Four families out of 6 answered “Yes,” while S1 and S5 families would not repeat the treatment.