Volume 35, Issue 8

Objectives To evaluate the effect of vitamin D supplementation on glycemic control in children and adolescents with T1DM. Content A systematic search was conducted of the Medline/PubMed, Web of Science, Embase, BVS/Lilacs, Cochrane Library, Scopus, Cinahl, Food Science, and FSTA databases. Two reviewers independently extracted article data and assessed quality. Summary A total of 1,613 eligible articles were retrieved, ten of which met the selection criteria: eight clinical trials, one retrospective cohort study, and one cross-sectional study. Regarding the cutoff points used to classify vitamin D status, most of the studies set deficiency at 25-hydroxyvitamin D <20 ng/mL, sufficiency at ≥30 ng/mL, and insufficiency as the interval between these values. Regarding intervention strategies, most used cholecalciferol for supplementation, but there was great variation in the dose and supplementation time. When evaluating the effect of vitamin D supplementation on HbA1c, a significant improvement in glycemic control was observed in 50% of the studies. However, only one of these studies was classified as being of positive methodological quality, with three having their quality classified as neutral and one as negative. Outlook There is yet no consistent evidence on the effect of vitamin D supplementation on glycemic control as an adjuvant in the treatment of children and adolescents with T1DM.

Objectives To explore the associations of environmental endocrine disruptors on precocious puberty in girls. Methods This was a case-control study in which 30 girls with precocious puberty and 46 age- and race-matched prepubertal females were enrolled. The concentrations of 10 environment endocrine disruptors (bisphenol A, bisphenol B, butylparaben, propylparaben, ethvlparaben, methylparaben, mono-butyl phthalate, mono-2-ethylhexyl phthalate, monoethyl phthalate, and monomethyl phthalate) in urine and 10 steroid hormones (dihydrotestosterone, corticosterone, hydrocortisone, 11-deoxycortisol, 17α-hydroxy progesterone, 4-androstene-3,17-dione, estrone, deoxycorticosterone, pregnenolone, and dehydroepiandrosterone) in serum were detected with the liquid chromatography-mass spectrometry (LC-MS). Results According to the Mann–Whitney U test, urinary levels of bisphenol A, monobutyl phthalate, and monomethyl phthalate were significantly higher in the precocious group than in the prepubertal group, and blood levels of hydrocortisone, 11-deoxycortisol, corticosterone, deoxycorticosterone, and pregnenolone were significantly lower in the precocious group than in the prepubertal group (p<0.05, VIP>1). Conclusions Our findings confirm the association between phthalate exposure and the incidence of precocious puberty in girls. Control and reduction of children exposure to phthalate esters should be considered as a health priority.

Objectives Fetal adrenals are one of the main organs responsible for maturation and survival. Extant literature is not clear about whether second trimester fetal adrenals are capable of synthesizing secreting aldosterone. Methods We have taken 20 adrenals from fetuses of intrauterine death cases. None of the fetuses had any external malformations and obstetric history was unremarkable. The organs were weighed and homogenized. The supernatant was used for aldosterone estimation by ELISA. Results We consistently detected aldosterone in all the cases including second trimester. However, we did not see any correlation between aldosterone concentration and gestational age. It is striking to note that there are wide variations in the tissue levels of aldosterone across different gestational ages and also same period. Conclusions Tissue aldosterone levels in second trimester can be possibly induced by stress preceding intrauterine deaths. It is possible that functional status of adrenal is different in intrauterine death cases as opposed to elective abortions in second trimester.

Objectives XLHR in children with a Rickets Severity Score ≥2 can now be treated with the new antibody drug, Burosumab, which prevents bone deformities and increases gait endurance. This study illustrates the extent of complications in the traditional orthopedic treatment of XLHR. The impact of surgery and severity of complications in this patient population has not been systematically assessed before. Methods The search strategy resulted in 215 studies and data were collected from 19 eligible studies and complications were categorized. Four medical charts of patients with XLHR at Aalborg University Hospital were assessed. Results One complication occurred on average per surgical procedure for XLHR in the published literature. The 168 reported complications were categorized as follows: Type I (n=79): Complications with minimal intervention required and treatment goal still achieved, Type II (n=41): Complications with substantial change in treatment plan and treatment goal still achieved, Type IIIA (n=23): Complications with failure to achieve treatment goal and no new pathology or permanent sequelae, Type IIIB (n=25): Complications with failure to achieve treatment goal and/or new pathology or permanent sequelae. Conclusions In average, one complication occurred per surgery and the severity of complications were substantial. The treatment goal was not achieved in 28% of surgeries whereof half of them resulted in permanent sequalae or new pathology. Our findings support the use of Burosumab for treatment of the skeletal changes in XLHR as the reported side-effects in Burosumab treatment appear negligible compared to the impact of surgeries and related complications (Imel EA, Glorieux FH, Whyte MP, Munns CF, Ward LM, Nilsson O, et al. Burosumab versus conventional therapy in children with X-linked hypophosphataemia: a randomised, active-controlled, open-label, phase 3 trial. Lancet 2019;393:2416–27). However, orthopedic surgery might still be needed for correcting deformities restricting activities of daily living in XLHR patients.

Objectives The role of insulin sensitivity (IS) in the development and progression of metabolic syndrome (MS) in subjects with type-1 diabetes (T1D) is being increasingly recognized. As patients with T1D lack endogenous insulin secretion, measurement of insulin concentration by immunoassay or by indices such as homeostasis model of assessment for insulin resistance (HOMA-IR) is not helpful in assessing IS. Hence, some equations have been developed and validated against data from euglycemic-hyper-insulinemic clamp tests (the gold standard) to estimate IS. 1) To assess IS using available equations (EDC, SEARCH and CACTI) and relationship of IS with MS and microalbuminuria in adolescents with T1D, (2) To compare the predictive value of these equations for detection of MS and derive a cut-off to predict the future risk of development of MS and microalbuminuria and (3) To identify the most accurate non-invasive and easy-to-use equation for detecting patients with double diabetes (DD) in a clinical setting. Methods This cross-sectional study included 181 adolescents aged 12–18 years with T1D. Demographic data and laboratory measurements were performed using standard protocols. IS was calculated using following equations:(1) EDC=24.31−12.22×(WHR)−3.29×(hypertension)−0.57×(HbA1c), (2) SEARCH=exp(4.64725−0.02032(waist)−0.09779(HbA1c)−0.00235(Triglycerides), (3)CACTI-exA=exp(4.1075–0.01299×(waist)−1.05819×(insulin dose)−0.00354×(Triglycerides)−0.00802×(DBP)). Results IS determined by all three methods had significant negative correlation (p<0.05) with MS as well as with microalbuminuria. The cut-off value of 5.485 mg/kg/min by SEARCH method for determining IS had the highest sensitivity and specificity in identifying MS. Conclusions IS by SEARCH equation may be used in routine clinical practice to detect DD in Indian adolescents with T1D at risk of developing metabolic as well as microvascular complications.

Objectives The growth and development of children is influenced not only by heredity factors but also by environmental factors, including nutrition and temperature. The aim of this study was to evaluate the growth and nutritional status of preschool children in Daxing’anling, the coldest region of China. Methods A descriptive, cross-sectional survey was performed among preschool children aged 3–6 years by stratified cluster sampling in Daxing’anling. The children’s parents completed the questionnaires. Height, body weight and head circumference were measured, and Z scores for weight for height, weight for age, height for age and head circumference for age were evaluated. Anthropometric data were compared with World Health Organization standards and China’s growth references. The levels of vitamin A, E and 25-(OH)-D 3 in serum were detected by high-performance liquid chromatography. Results A total of 305 children were recruited. The average height of the preschool children was lower than China’s growth reference but higher than the WHO standard. More than half of the preschool children ranged from −1 SD to +1 SD. Both the values of weight for height and of weight for age were positive and higher than the WHO standards (p<0.01), with a significant difference between boys and girls (p<0.01). The incidences of stunting, wasting, and underweight were 4.59%, 2.95%, and 2.30%, respectively, although the prevalence of overweight and obesity was high (18.03% and 6.89%, respectively). The rates of vitamin A and D deficiency were 7.54% and 88.85%, respectively. Vitamin A was also positively associated with 25-(OH)-D 3 . Conclusions The burden of malnutrition in preschool children exists in cold regions, and a cold climate may be an important factor. Therefore, we should pay attention to the nutrition and physical growth of local preschool children; in particular, vitamin D deficiency should be given high priority, and necessary nutritional interventions should be made.

Objectives Coexistence of arterial hypertension (AH) in children with obesity increases morbidity and shortens life. Its role as an indicator of coexisting metabolic complications is however less known. The objective of the study was to compare metabolic profiles of children with obesity and with or without AH. Methods We included patients aged 10–18 with the BMI Z-score ≥2. Diagnosis of AH was based on the European Society of Hypertension criteria (2016). Metabolic profiles were assessed by glucose and insulin levels taken before and after glucose load, fasting levels of triglycerides (TG), total (TC), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, and HOMA-IR. Results Of 534 patients, 33.5% were diagnosed with AH. The AH patients, as compared to non-AH, had higher fasting insulin levels (22 vs. 19.7 mIU/L, p=0.04), HOMA-IR (4.5 vs. 4.0, p=0.029), and post-load glucose level (6.3 vs. 5.7, p=0.000041). No differences in the post-load insulin levels (113 vs. 100 mIU/L, p=0.056), fasting glucose (4.5 vs. 4.5 mmol/L, p=0.5), or lipids were found (TC: 4.4 vs. 4.4 mmol/L, p=0.9; LDL: 2.7 vs. 2.7, p=0.2; TG: 1.4 vs. 1.4 mmol/L, p=0.5; HDL: 1.1 vs. 1.2, p=0.3. Conclusions Concomitance of AH in children with obesity may be an indicator of coexisting metabolic complications.

Objectives To verify the associations between prenatal and perinatal factors with offspring body mass index (BMI) and the moderator role of maternal BMI in this relationship. Methods Cross-sectional study developed with 1,562 children and adolescents aged between 6 and 17 years, as well as their mothers, from southern Brazil. The prenatal and perinatal factors, weight, and height for the calculation of maternal BMI were self-reported. For the calculation of BMI, weight and height of the child/adolescent were measured on an anthropometric scale with a coupled stadiometer. Linear regression models were used for the moderation analysis. All analyzes were adjusted for the mother’s and child’s age, sex, sexual maturation, skin color/race, and educational level. Results cesarean as type of delivery (β=0.66; 95% CI=0.22 1.04; p=0.002) and pregnancy complications (β=0.60; 95% CI=0.15 1.04; p=0.002) were positively associated with offspring BMI. Schoolchildren who were breastfed for 4–6 months showed −0.56 kg/m 2 of BMI (95% CI=−1.06–0.06; p=0.02). Birth weight was also associated with BMI, with low weight being inversely (β=−0.59; 95% CI=−1.03–0.15; p=0.008), while overweight was positively related (β=0.84; 95% CI=0.08 1.60; p=0.02). The moderation analysis indicated a positive interaction between the mother’s BMI and cesarean, pregnancy complications, and smoking with the offspring’s BMI. On the other hand, there was an inverse association between breastfeeding from 7 to 12 months and the offspring BMI, only in mothers with high BMI. Conclusions Adequate maternal BMI is essential to prevent a high BMI in their children, especially when considering the influence of prenatal and perinatal risk factors.

Objectives Non-syndromic monogenic obesity is a rare cause of early-onset severe obesity in the childhood period. The aim of this study was to screen four obesity related genes ( LEP, LEPR, MC4R and POMC ) in children and adolescents who had severe, non-syndromic early onset obesity. Methods Next-generation sequencing of all exons in LEP, LEPR, MC4R and POMC was performed in 154 children and adolescents with early onset severe obesity obesity. Results Fifteen different variants in nineteen patients were identified with a variant detection rate of 12.3%. While six different heterozygous variants were observed in MC4R gene (10/154 patients; 6.5%), five different variants in POMC gene (four of them were heterozygous and one of them was homozygous) (6/154 patients; 3.9%) and four different homozygous variants in LEPR gene (3/154 patients; 1.9%) were described. However, no variants were detected in the LEP gene. The most common pathogenic variant was c.496G>A in MC4R gene, which was detected in four unrelated patients. Six novel variants (6/15 variants; 40%) were described in seven patients. Four of them including c.233C>A and c.752T>C in MC4R gene and c.761dup and c.1221dup in LEPR gene were evaluated as pathogenic or likely pathogenic. Conclusions In conclusion, MC4R variants are the most common genetic cause of monogenic early-onset obesity, consistent with the literature. The c.496G>A variant in MC4R gene is highly prevalent in early-onset obese patients.

Objectives We aimed to evaluate the association between vitamin B12, folate, homocysteine levels, and carotid intima-media thickness (CIMT) among children with obesity in whom vitamin deficiencies are more frequent. Methods Herein, 100 children with obesity (58 girls) were included (age, 5–18 years). Height, weight, body mass index (BMI), waist circumference (WC), puberty stage, blood pressure, and biochemical values were collected from medical records; standard deviations (SDS) and percentiles were calculated. Obesity was defined as BMI SDS of >+2SDS. Vitamin B12 and folate levels of <300 pg/mL and <4.8 ng/mL, respectively, were considered deficient. A radiologist quantified measurements from the carotid artery. Results Mean patient age was 12.52 ± 3.63 years. The mean weight SDS, BMI SDS, and WC/height were +3.37 ± 0.93, +2.93 ± 0.55, and 0.65 ± 0.05, respectively. In pubertal cases, insulin (p<0.001), the homeostatic model assessment for insulin resistance (HOMA-IR) (p=0.001) and homocysteine (p=0.002) levels were higher; vitamin B12 (p<0.001) and folate (p<0.001) levels were lower than those in prepubertal ones. WC and HOMA-IR correlated with CIMT; however, homocysteine levels were not correlated with CIMT. Conclusions In our study, pubertal cases had lower vitamin B12 and folate levels as well as higher homocysteine levels. Although no correlation was identified between homocysteine levels and CIMT, this condition may be related to our study group comprising children, who had a shorter duration of obesity than those in adults. As CIMT was higher in children/adolescents with increased WC, it is proposed that they need central obesity more frequently and carefully follow-up.

Objectives Growth hormone deficiency (GHD) in adults is associated with an increased risk of cardiovascular morbidity and mortality. Although children with GHD are also believed to have a similar cardiovascular disease (CVD) risk beginning at an early age, the available data in children is scarce. We aimed to determine the various CVD risk parameters in children with isolated GHD (IGHD). Methods A cross-sectional case-control study was conducted at a tertiary care centre in North India comparing various auxological, biochemical, and echocardiographic parameters between 20 IGHD children aged 5–15 years and their age and sex-matched healthy controls. Results The mean age of children with IGHD and controls was similar (10.5 ± 2.6 yr vs. 9.9 ± 2.7 yr, p=0.48). Children with IGHD had significantly higher waist-hip-ratio (p=0.01), total cholesterol (p=0.02), non-high-density lipoprotein–cholesterol (p=0.02), serum homocysteine (p<0.001), C-reactive protein (CRP) (p=0.01) and pro-brain natriuretic peptide (pro-BNP) (p=0.04) levels as compared to healthy controls. Left ventricular mass (LVM) and interventricular septal thickness were significantly lower (p=0.04; p=0.02) in IGHD children. Correlation analysis showed that pro-BNP and CRP levels had negative correlation (p<0.001, r=−0.70; and p=0.04, r=−0.44, respectively) and LVM had a positive correlation (p=0.02, r=0.53) with height SDS among IGHD children. Conclusions Children with IGHD showed abnormalities in several biochemical and cardiac parameters that may be associated with an increased CVD risk in later life. More extensive studies, including younger children with IGHD, are needed to determine the lower ages at which the CVD risk is detectable.

Objectives To evaluate the relationships between adipose tissue distribution, insulin secretion and sensitivity, sleep-disordered breathing, and inflammation in obese adolescents. Methods Cross-sectional study of 56 obese adolescents who underwent anthropometric measures, dual-energy X-ray absorptiometry, overnight polysomnography, oral glucose tolerance test (OGTT) and frequently sampled intravenous glucose tolerance test. Correlation and regression analyses were used to assess relationships between adiposity, insulin secretion and sensitivity, measures of sleep-disordered breathing (oxyhemoglobin nadir, SpO 2 ; apnea hypopnea index, AHI; arousal index, AI; maximum end-tidal CO 2 ; non-REM sleep duration), and inflammation (high-sensitivity C-reactive protein, hsCRP). Results Subjects (55% female) were mean (SD) 14.4 (2.1) years, with BMI Z-score of 2.3 (0.4). AHI was >5 in 10 (18%) subjects and 1< AHI ≤5 in 22 (39%). Visceral adipose tissue area (VAT) was positively correlated with OGTT 1 and 2 h insulin and 1 h glucose, and hsCRP (r=0.3–0.5, p≤0.007 for each). VAT was negatively correlated with sensitivity to insulin (r=−0.4, p=0.005) and SpO 2 nadir (r=−0.3, p=0.04) but not with other sleep measures. After adjustment for BMI-Z, sex, population ancestry, age, and sleep measures, VAT remained independently associated with insulin measures and 1 h glucose, but no other measures of glycemia. SAT was not associated with measures of glycemia or insulin resistance. Conclusions Among adolescents with obesity, visceral adiposity was associated with insulin resistance, SpO 2 nadir, and inflammation. The independent association of visceral adiposity with insulin resistance highlights the potential role of VAT in obesity-related chronic disease.

Objectives Post-traumatic brain injury hypopituitarism is a common unrecognized condition in children after head injury. Due to its similarity of clinical symptoms with those of head trauma, clinical diagnosis of post-TBI hypopituitarism is challenging. To date, there is no standardized screening protocol for children with history of brain injury. This article demonstrates a case of 14-year-old boy with severe head trauma who developed refractory seizures with episodic hypoglycemia and weight loss. We aimed to focus on the prevalence, clinical courses and clinical implementations of each hormonal axis in children with post-traumatic brain injury hypopituitarism. We also aim to raise awareness of this condition to pediatricians in light of enhancing patient care. Methods We have searched for original articles, published in English between year 2000 and 2021. There are 20 related articles, authors reviewed all the articles independently. Results Prevalence of post-traumatic hypopituitarism ranges from 5–57% in children. Growth hormone is the most commonly affected hormone. The highest prevalence is 42.3% at more than 12 months after the brain injury. The symptoms and severity range from asymptomatic to requiring long-term hormonal therapy. Although normalization of pituitary function is demonstrated at various times after the injury, hormone replacement therapy is still required in some patients. Conclusions This is the first report that demonstrates a presenting symptom of hypopituitarism mimic traumatic brain symptoms which result in it being overlooked. This case emphasizes the need to develop pituitary function screening protocols for children with TBI. We have proposed our pituitary screening protocol for children with TBI in this article.

Objectives Central diabetes insipidus (CDI) is a rare but important disease of varying etiology that poses challenges in diagnosis and follow-up. Identifying diagnostic difficulties in patients with CDI will help ensure an optimal approach to their management and follow-up. This study aimed to characterize the clinical and etiological characteristics of CDI in pediatric patients. Methods We analyzed the admission and follow-up data of CDI patients aged 0–18 years who were followed in our center between 2010 and 2019. Results The study included 56 patients with a mean age at diagnosis of 7.92 ± 5.11 years and symptom duration of 8.65 ± 21.3 months. The patients were grouped by etiology into those with organic causes, such as structural anomalies, tumors, and trauma (group 1, n=41) and other causes (group 2, n=15). The prevalence of idiopathic CDI was 16%. At least one pituitary hormone deficiency was detected in 60.7%, the most common being thyroid stimulating hormone deficiency. Patients in group 1 had a higher mean age at diagnosis, shorter symptom duration, and higher frequency of other pituitary hormone deficiencies compared to group 2. Additionally, germinoma was detected 1 year subsequent to normal MRI findings at diagnosis and another patient was diagnosed with Langerhans cell histiocytosis (LCH) 5 years after diagnosis. All patients responded well to replacement therapies, but two patients with germinoma died during follow-up. Conclusions In the pediatric age group, intracranial organic pathologies are an important etiology of CDI, and despite a short symptomatic period, determining the cause may be challenging and prolonged. Patients presenting at a young age with a long history of symptoms and no other pituitary hormone deficiency are unlikely to have organic CDI. However, organic causes such as LCH should be evaluated at all ages. Patients with idiopathic disease are candidates for further etiological studies, and repeated cranial imaging is important during follow-up.

Objectives Hypochondroplasia (HCH) is characterized by disproportionate short stature and regarded as a milder form of achondroplasia (ACH), which is another skeletal dysplasia, both caused by variants in fibroblast growth factor receptor 3 ( FGFR3) gene. HCH diagnosis is based on the clinical features and skeletal survey findings. The most common FGFR3 variant in HCH affects the codon 540, leading to substitution of asparagine with lysine in about 70% of patients. Case presentation Herein, we described the clinical and radiographical manifestations of HCH in affected members of a Turkish family with very rare Asn540Thr (c.1619A>C) variant within hot spot of the gene for this condition. Conclusions This is a very rarely reported variant in the literature and this report is the first case with this variant in Turkish population. The report also presents the phenotypic variability within a family with the same variant, which is inherent to HCH.

Objectives Rapid-onset obesity with hypoventilation, hypothalamic dysfunction, autonomic dysregulation, and neural-crest tumour (ROHHAD(NET)) is a rare syndrome presenting in early childhood associated with high morbidity and mortality. There is no specific diagnostic biomarker and diagnosis is based on clinical features. An autoimmune origin has been postulated. Case presentation Management is largely supportive. We report a case of a five-year old female who presented in respiratory arrest after 6-months of rapid weight gain. She had central hypoventilation, central diabetes insipidus, growth hormone deficiency and hyperprolactinaemia. She displayed elevated interleukin-6 levels on cytokine serology which normalised after rituximab treatment. After rituximab treatment, her weight reduced significantly from greatly above the 99.6th to the 50th centile in 12 months. Conclusions This response possibly reflects an underlying, immune-inflammatory pathology driving excess adiposity in this condition. Potentially, other aspects of ROHHAD(NET) may be mediated through autoimmune dysregulation in which case rituximab may provide benefits for prognosis and survival.

Objectives Ectopic parathyroid hormone (PTH) secretion is rare in children with rhabdomyosarcoma, and only a few pediatric cases have been reported to date. Reports of the use of zoledronic acid (ZA) and Denosumab are limited for the treatment of hypercalcemia of malignancy (HCM) in the pediatric population. The aim of presenting this pediatric case of rhabdomyosarcoma accompanied by HCM, secondary to ectopic PTH secretion, was to highlight the benefits of ZA as a first-choice bisphosphonate in this situation with Denosumab as an alternative therapy. Case presentation The patient was diagnosed at 13 years with alveolar rhabdomyosarcoma. Multiple bone metastases first appeared at 15 years, but he remained normocalcemic until 17 years old when serum calcium was 15.1 mg/dL and PTH 249 pg/mL. While serum calcium responded well after ZA and Denosumab cycles, PTH remained elevated, reaching a peak value of 1851 pg/mL during treatment cycles. Conclusions We report a patient with rhabdomyosarcoma accompanied by HCM, secondary to ectopic PTH, in whom the HCM was successfully managed with ZA and Denosumab. We believe that ZA should be the bisphosphonate of choice in pediatric HCM with rhabdomyosarcoma, while Denosumab may be another option in ZA-refractory cases.

Objectives Niemann–Pick disease type A (NPDA, MIM: 257200) is an autosomal recessive sphingolipidosis caused by lysosomal acid sphingomyelinase (ASM) deficiency. A cluster of genes located at chromosome 11p15 have been reported to be imprinted genes, such as TSSC5, TSSC3, and ZNF215 that flanking SMPD1 gene. It was reported by a few recent studies that SMPD1 gene was paternally imprinted and maternally preferentially expressed. Case presentation A five-month-old boy with severe anemia, hepatosplenomegly and bone marrow foam cells was recruited from a complete cousin couple. To determine whether boy suffered from NPDA, ASM activity and SMPD1 gene sequencing were performed on available individuals of this pedigree including the proband, his parents and sister. The ASM activities of proband and parents showed deficiency (17.7 nmol/h/g-protein) and about 50% decreased (83.3 nmol/h/g-protein), respectively, compared with normal controls (204.5 nmol/h/g-protein). SMPD1 gene sequencing in the proband revealed a homozygous mutation c.1420_1421del, which leads to an open reading frameshift and a premature stop codon. The parents and some individuals of this family demonstrated heterozygous mutation at this locus. To investigate whether SMPD1 gene is imprinted as reported previously, the expression of RNA level was studied in the whole family members available. The members with heterozygous mutation for c.1420_1421del showed that both paternal and maternal inherited alleles were expressed. Conclusions This study reported a c.1420_1421del mutation in SMPD1 gene which caused ASM activity decrease and this locus was biallelically expressed in heterozygous subjects implicating SMPD1 is not imprinted in this family.