Lipid profile in Noonan syndrome and related disorders: trend by age, sex and genotype

Background RASopathies are developmental disorders caused by dysregulation of the RAS-MAPK signalling pathway, which contributes to the modulation of multiple extracellular signals, including hormones and growth factors regulating energetic metabolism, including lipid synthesis, storage, and degradation. Subjects and methods We evaluated the body composition and lipid profiles of a single-centre cohort of 93 patients with a molecularly confirmed diagnosis of RASopathy by assessing height, BMI, and total cholesterol, HDL, triglycerides, apolipoprotein, fasting glucose, and insulin levels, in the context of a cross sectional and longitudinal study. We specifically investigated and compared anthropometric and haematochemistry data between the Noonan syndrome (NS) and Mazzanti syndrome (NS/LAH) groups. Results At the first evaluation (9.5 ± 6.2 years), reduced growth (-1.80 ± 1.07 DS) was associated with a slightly reduced BMI (-0.34 DS ± 1.15 DS). Lipid profiling documented low total cholesterol levels (< 5th percentile) in 42.2% of the NS group; in particular, in 48.9% of PTPN11 patients and in 28.6% of NS/LAH patients compared to the general population, with a significant difference between males and females. A high proportion of patients had HDL levels lower than the 26th percentile, when compared to the age- and sex-matched general population. Triglycerides showed an increasing trend with age only in NS females. Genotype-phenotype correlations were also evident, with particularly reduced total cholesterol in about 50% of patients with PTPN11 mutations with LDL-C and HDL-C tending to decrease during puberty. Similarly, apolipoprotein A1 and apolipoprotein B deficits were documented, with differences in prevalence associated with the genotype for apolipoprotein A1. Fasting glucose levels and HOMA-IR were within the normal range. Conclusion The present findings document an unfavourable lipid profile in subjects with NS, in particular PTPN11 mutated patients, and NS/LAH. Further studies are required to delineate the dysregulation of lipid metabolism in RASopathies more systematically and confirm the occurrence of previously unappreciated genotype-phenotype correlations involving the metabolic profile of these disorders.


Introduction
Dysregulation of the RAS-MAPK pathway contributes to the control of metabolism and energy storage (1). Energy metabolism is consistently found to be dysregulated in RASopathies (2)(3)(4)(5)(6), which constitute a family of disorders caused by mutations in genes encoding key transducers participating in the RAS-MAPK signalling cascade (7,8). Metabolic and nutritional aspects in RASopathies are, however, still poorly explored (9). Feeding difficulties are well recognized to represent a relevant concern in these disorders, especially during infancy and in association with specific genotypes (10, 11). A reduction of adiposity has been documented in patients with Noonan syndrome (NS; MIM: PS163950) (12) and LEOPARD syndrome (also known as Noonan syndrome with multiple lentigines) (NSML; MIM: PS151100) (13) and a particularly low BMI score has been reported in Mazzanti syndrome (also known as Noonan-like disorder with loose anagen hair) (NS/LAH; MIM: 607721) (14). A recent study on body composition in RASopathies suggests the occurrence of simultaneous impairment in adipose and muscle mass tissues (9).
Few data are available on the metabolic and lipid profiles characterizing these disorders. Based on these considerations, the aim of this work was to evaluate the fasting lipid profile, in particular cholesterol and triglycerides levels, in a large and unselected single-centre cohort of patients affected by a RASopathy with a molecularly confirmed diagnosis, who had been in follow-up from childhood until early adulthood.

Patients and methods
A retrospective longitudinal study was performed involving 93 patients with clinical features fulfilling the criteria for RASopathies and whose diagnosis had been confirmed by molecular analysis.
The patients were recruited at the Paediatric Rare Disease Outpatient Unit of IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy, from 2001 to 2020, and were followed until April 2022. Molecular analyses were performed by parallel sequencing using a continuously updated panel of genes implicated in these disorders, as previously reported (14). For a subset of patients, the molecular diagnosis had been achieved by means of targeted Sanger sequencing.
A fasting blood sample was taken every six months or yearly from each patient. The following parameters were analyzed: total cholesterol (TC), HDL cholesterol (HDL-C), triglycerides (TG). LDL cholesterol (LDL-C) blood levels were calculated using the Friedewald formula (LDL cholesterol = total cholesterol -HDL cholesteroltriglycerides/5) (15).
Among the different RASopathies belonging to this cohort, here we present, discuss and compare the anthropometric and haematochemistry data collected in the NS and the NS/ LAH groups.
The study was conducted according to the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by the Emilia-Romagna AVEC ethics committee [internal code 119/ 2022/Oss/AOUBo].

Statistical analysis
Height and body mass index (BMI, Kg/m 2 ) were expressed as standard deviation scores (SDS) and referred for age-and sexspecific groups. Anthropometric measurements were compared to the standard growth curves for the general Italian population (16).
Blood concentrations of TC and lipoproteins in patients between 12 and 20 years of age were compared to the corresponding age and sex standards of the "National Health and Nutritional Examination Surveys" (17). Specifically, we used the general population 5 th percentile as a reference point for low levels of TC and LDL, the 26 th percentile for low levels of HDL-C, and the 54 th percentile for above normal values of LDL.
The distributions of lipid profile biomarker levels (TC, HDL-C, LDL-C and TG) were compared across genotypes using the Mann-Whitney test. The trajectories of those biomarkers for age and sex were obtained as margins (linear predictions) from regression models in which the biomarker was the dependent variable, and the interaction of sex and age (in rounded years) was the predictor. Data collected at all ages were used in these regressions, and standard error correction was included to account for repeated data from the same patients.
The proportions of biomarkers with values below or above the standard cutoffs were compared across sexes and genotypes using chi-square or Fisher's exact test.
All analyses were performed using Stata v.17.0; p-values lower than 0.05 were considered statistically significant.

Results
The study cohort included 93 patients, 53 (57.0%) males and 40 (43.0%) females; at the first evaluation mean age was 9.5 ± 6.  Table 1). Among the NS patients, 49 (70.0%) had PTPN11 mutations, while 21 patients were non-PTPN11 and each of the other genotypes accounted for less than 10%. In the NS/LAH group of patients, 9 out of 10 had a SHOC2 mutation, the remaining having a pathogenic missense change in PPP1CB.

Lipid profile
Considering all the blood samples collected in the NS and NS/ LAH patient groups, TC levels were significantly higher in females than males (Table 2): 156.0 ± 31.2 vs. 137.6 ± 29.5 mg/dL (p<0.001) in NS patients and, in particular, 152.5 ± 32.6 vs. 136.1 ± 27.1 mg/ dL (p=0.001) in the NS subgroup with mutated PTPN11, and 160.8 ± 34.3 vs. 133.5 ± 18.7 mg/dL (p=0.013) in NS/LAH patients. Also, LDL-C and TG were higher in females, while HDL-C values were similar for both sexes ( Table 2).
The estimated trajectories of TC, HDL-C and LDL-C decreased with age in NS patients, with similar trends seen in both males and females. In NS/LAH, values decreased with age in males and were stable or slightly increased in females, although few subjects were evaluated. Triglycerides showed an increasing trend with age in NS females and a stable trend in NS males and in NS/LAH (Figure 1).
When comparing our patients affected by NS and NS/LAH with the general population, we tested all the blood samples collected in the 12 to 20-year age range ( Table 3).
The lipid profile was characterized by TC values lower than the 5 th percentile for the general population in 42.2% of the NS group (41.3% in males vs 44.4% in females, p=0.819); in particular, in 48.9% of the PTPN11-related NS subgroup (46.7% in males vs. 53.3% in females, p=0.673), in 26.3% of the non-PTPN11 NS subgroup (31.2% in males vs. 0% in females, p=0.530), and in 28.6% of NS/LAH patients (33.3% in males vs. 25.0% in females, p=0.594) (Figure 2).
Females showed a significantly higher percentage of above normal LDL-C levels than males when considering the NS and NS/LAH groups (34.6% vs 11.5%, p=0.015). The proportions of low  Model-estimated mean values and 95% CI of lipid profile biomarkers (TC, HDL-C, LDL-C and triglycerides) by age (1-20 years) and sex, for NS and NS/LAH patients.  (Table 3). When comparing the lipid profiles of the different genotypes, NS patients showed higher levels of HDL-C than NS/LAH patients (p=0.046) and a higher ratio of HDL/LDL (p=0.035) ( Table 4).

Lipid profile and puberty
Lipid profiles were obtained from assessments of 90 prepuberal females (mean age 11.6 ± 8.5), 116 prepuberal males (mean age 9.6 ± 6.2), 29 pubertal females (mean age 17.6 ± 4.7) and 61 pubertal males (mean age 17.5 ± 4.9). Significant differences between prepubertal and pubertal assessments, with lower values in puberty, were found in males for TC (127.2 vs. 141.7 mg/dl in puberty vs. pre-puberty,  Distribution of age-and sex-related values of TC, LDL-C and HDL-C, compared with increased-risk curves for the general population (age 12-20 years) taken from Joliffe C.J. and Janssen I (17). The 5 th percentile was the reference point for low levels of TC and LDL, the 26 th percentile for low levels of HDL-C, and the 54 th percentile for above normal values of LDL. However, no significant differences between prepubertal and pubertal assessments was found in the proportion of subjects with TC, LDL-C, HDL-C and TG values below the lowest threshold. The same pattern of significantly lower TC and LDL-C values in pubertal compared to prepuberal assessments was found also when analysing NS patients and NS/LAH separately. In NS patients, a significant difference was also found for HDL-C, again with lower values in puberty (42.4 vs. 47.6 mg/dl, p=0.005).

Glucose metabolism
Blood glucose levels were nearly significantly higher in NS patients than in NS/LAH subjects (mean values: 80.7 vs. 76.0, p = 0.060). HOMA IR levels did not differ significantly (1.47 vs. 1.37, p = 0.758). In both groups, the mean values were within the normal range for the general population.

Discussion
In this study, we characterize the lipid profile in a large and unselected cohort of patients with RASopathies.
In the general population, a physiological fluctuation in lipoprotein levels during adolescence and young-adult age has been reported. In particular, Jolliffe and Janssen (17) elaborated curves corresponding to borderline high and high levels for TC and LDL and for low and protective HDL-C. Our data provide evidence that patients with NS and NS/LAH show significant differences in their lipid profile compared to the general population in the 12 to 20-year age range, according to age and sex. Specifically, TC values were lower than the 5 th percentile for the general population standard range in 42.2% of the NS group (in particular, in 48.9% of patients carrying a mutated PTPN11 allele) and in a lower percentage of the NS/LAH group (28.6%).
In relation to the values trend for age and gender, Joliffe and Janssen (17) reported that in both sexes TC concentration declines during the early pubertal period and subsequently increases to reach adult levels, while LDL-C and HDL-C present a different trajectory in males and females. In males, LDL-C decreases in early adolescence and then increases from 15.5 years of age, instead, in females, there is a regular increase until adulthood. HDLconcentration in males declines moderately until 16 years of age and then stabilizes, whereas females do not show any modification during the pubertal period.
Even in our cohort, lipid profiles differed by sex, as females had significantly higher TC, LDL-C and TG levels than males in all the groups of patients evaluated, although they remain within the normal range for the general population. As shown in Figure 1, in puberty, differently from what is seen in the GP, TC and LDL-C values were lower than those collected in prepuberal age in males, also when analyzing the NS and NS/LAH groups. In females, this trend was confirmed only in the NS group. Moreover, NS patients also HDL-C showed lower values in puberty.
Our cohort seems to have a lipid profile characterized by low TC values, as seen in about 50% of the NS patients with PTPN11 mutation, low HDL levels, as seen in NS patients (in particular in PTPN11 mutated patients), with LDL-C and HDL-C tending to decrease during puberty, more evidently in males. The temporal patterns of lipid profile were quite opposed for NS and NS/LAH, with the former generally displaying decreasing values (except for triglycerides) while NS/LAH, especially among females, showed increasing trends with age. Triglycerides showed an increasing trend with age only in NS females. We did not observe any pathological involvement glucose homeostasis in our patients.
A slightly lower BMI was also observed, without correlation to TC and cholesterol fractions. It is currently known that low BMI is a typical feature of RASopathies and overweight has a very low prevalence, probably due to the primary involvement of adipose tissue and muscle mass (9,12).
Previous studies in patients with RASopathies have shown that body composition changes are not a reflection of the contribution of caloric and macronutrient intake when evaluating dietary habits and energy expenditure, because the results were similar to those of the general population (9).
Currently, in the literature, there are no studies that allow us to clarify the pathogenesis of the lipid profile in RASopathies, although the dysregulation of RAS-MAPK pathway in adipogenesis has been demonstrated. In fact, studies in cellular models and knockout mice revealed an important role for ERK1 and SHP2 in adipogenesis, resulting in defective lipid metabolism (2). Our results seem to support this hypothesis: in fact, patients with PTPN11 mutations have shown greater involvement of the lipid profile, with high prevalence of low lipid levels, compared to the general population.
Further studies on in vitro and in vivo experimental models are required to understand the role of the RAS-MAPK pathway in regulating lipid metabolism and how alterations in this pathway affect metabolic profiles. It will also be important to evaluate whether the global metabolic imbalance can influence other characteristic features of RASopathies such as growth, delayed puberty, heart disease, and cognitive impairment.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Ethics statement
The studies involving human participants were reviewed and approved by Emilia-Romagna AVEC ethics committee [internal code 119/2022/Oss/AOUBo]. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

Author contributions
FT, LM, ES, MT and AnP designed, interpreted the data, and wrote the manuscript. MS, AM and CS collected the data. DG and MZ provided the statistical analysis. FT, ES, and AnnP cared for the patients and coordinated all clinical investigation. CR carried out molecular analysis.

Funding
MT is recipient of EJP-RD funding (NSEuroNet).