Diagnosis of Chromosome 15q-Terminal Deletion Syndrome through Elevated Fasting Serum Growth Hormone Levels

: Chromosome 15q26-qter deletion syndrome is a rare disease that causes prenatal and postnatal growth retardation, microcephaly, developmental delay, and congenital heart diseases, mainly due to haploinsufﬁciency of IGF1R . In addition, patients with pathogenic variants of the IGF1R show similar symptoms. We report the case of a 5-month-old girl with prenatal and postnatal growth retardation, microcephaly, and congenital heart disease. At 5 months of age, her length was 54.7 cm ( − 4.3 SD), her weight was 4.4 kg ( − 3.1 SD), and her head circumference was 37.4 cm ( − 2.8 SD), thus presenting severe growth retardation. Repeated pre-feeding serum GH levels were abnormally high (26.1–85.5 ng/mL), and IGF-1 levels (+0.16 to +1.2 SD) were relatively high. The 15q sub-telomere ﬂuorescence in situ hybridization analysis revealed a heterozygous deletion in the 15q terminal region. Whole-genome single nucleotide polymorphism microarray analysis showed a terminal deletion of 6.4 Mb on 15q26.2q26.3. This is the ﬁrst report showing that fasting GH levels are high in early infancy in patients with IGF1R abnormalities. In addition to relatively high IGF-1 levels, elevated fasting GH levels in early infancy may contribute to the diagnosis of IGF1R abnormalities.


Introduction
Chromosome 15q-terminal deletion syndrome (OMIM #612626) is a heterozygous deletion of chromosome 15q26-qter that causes prenatal and postnatal growth retardation, microcephaly, developmental delay, and various abnormalities [1]. Several genes in the 15q26 region, such as insulin-like growth factor 1 receptor (IGF1R) and nuclear receptor subfamily 2 group F member 2 (NR2F2), show different phenotypes resulting from heterozygous deletions. Heterozygous deletion of the IGF1R is associated with intrauterine growth retardation and postnatal growth deficiency, minor facial anomalies including microcephaly, micrognathia and triangular faces, and skeletal anomalies such as cubitus valgus, clinodactyly and brachydactyly [2]. Heterozygous deletion of the NR2F2 is associated with congenital heart diseases (CHD), such as ventricular septal defect (VSD), atrial septal defect (ASD), coarctation of aorta, patent ductus arteriosus, and pulmonary stenosis [3,4]. The deletion of other genes in the 15q26 region may contribute to other phenotypic differences.
Since insulin-like growth factor 1 (IGF-1) and its receptor play an important role in skeletal growth and brain development, IGF1R deletions cause intrauterine and postnatal growth retardation, and mild developmental delay [5]. The binding of IGFs to its receptor results in the autophosphorylation of intracellular tyrosine residues, which activates the phosphatidylinositide-3-kinase (PI3K)/AKT, mitogen-activated protein kinase (MAPK) /extracellular signal-regulated kinase (ERK) signaling pathways. This, in turn, stimulates cell proliferation and growth [6].
Similarly, several patients with pathogenic variants of IGF1R, mainly in the heterozygous state and rarely in the compound heterozygous state, have also been reported to exhibit growth retardation before and after birth [7,8]. Short children born smallfor-gestational age (SGA) have been reported to possibly include patients with IGF1R pathogenic variants [9]. Indeed, variable phenotypic expression has already been reported, even in relatives carrying the same molecular defect, which makes the diagnosis of IGF1R defects difficult [8].
Although relatively high serum IGF-1 levels have been reported in patients with IGF1R abnormalities, the serum growth hormone (GH) levels in these patients remain unknown. We report the case of a girl with pre-and post-natal severe growth retardation, microcephaly, and ASD, where high serum GH levels in addition to relatively high IGF-1 levels led to a definitive diagnosis.

Case Presentation
The female patient presented with severe fetal growth retardation at 19 weeks of gestation. Fetal echocardiography revealed pericardial effusion and septal hypertrophy. She was delivered at 38 weeks of gestation by emergency cesarean section due to fetal distress. Her father's height was 174 cm, and her mother's height was 156 cm. Her birth weight was 2034 g (−2.6 SD), body length was 42 cm (−3.2 SD), and head circumference was 30.5 cm (−1.9 SD); she was born SGA. She had no skeletal abnormalities or dysmorphic facial features. Echocardiogram revealed an ASD type II and VSD. At one week of age, she presented with inspiratory stridor due to laryngomalacia, and required a high-flow oxygen nasal cannula to maintain oxygenation. In addition, nasogastric tube feeding was required due to feeding difficulties until 2 months of age. At 5 months of age, her length was 54.7 cm (−4.3 SD), her weight was 4.4 kg (−3.1 SD), and her head circumference was 37.4 cm (−2.8 SD). She showed delayed motor development, achieved head control at 5 months of age and could not rollover. On the other hand, her social and language skills were not delayed: her social smile was noted at 3 months of age and babbling at 4 months. The patient showed severe growth retardation; thus, she was referred to a pediatric endocrinologist. Repeated pre-feeding serum GH levels were abnormally high, and serum IGF-1 levels were slightly above the age-average levels ( Table 1). Although the blood glucose level decreased at 3 months of age, the fasting blood glucose remained at the lower limit of normal between 60 and 90 mg/dL. No severe hypoglycemia was observed during the first 3 months, and after 3 months of age, blood glucose levels were in the normal range. Other tests, such as renal function, liver function, metabolic markers, thyroid function, and chromosomal G-band test also did not reveal any abnormalities. Abdominal ultrasonography showed no morphological abnormalities of the kidneys, and head MRI showed no structural abnormalities of the brain.

Diagnostic Assessment: Genetic Testing
We first suspected IGF1R deletion based on the severe growth retardation before and after birth, CHD, high GH levels, and relatively high IGF-1 levels, and performed subtelomere fluorescence in situ hybridization (FISH) analysis focusing only on 15q. The 15q subtelomere FISH analysis revealed a heterozygous deletion in the 15q terminal region ( Figure 1). Whole-genome single nucleotide polymorphism (SNP) array analysis using the Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA) showed a terminal deletion of 6.4 Mb on chromosome 15q26.2-26.3, which contains diseasecausing OMIM genes, NR2F2, IGF1R, MEF2A, ADAMTS17, CERS3, LINS1, ALDH1A3 and CHSY1, and 1.7 Mb duplication on 15q26.2 which does not contain the disease-causing OMIM gene ( Figure 2). No other pathogenic copy number variation or copy number neutral loss of heterozygosity were observed in the SNP array. We diagnosed her with chromosome 15q26-qter deletion syndrome.

Diagnostic Assessment: Genetic Testing
We first suspected IGF1R deletion based on the severe growth retardation before and after birth, CHD, high GH levels, and relatively high IGF-1 levels, and performed subtelomere fluorescence in situ hybridization (FISH) analysis focusing only on 15q. The 15q subtelomere FISH analysis revealed a heterozygous deletion in the 15q terminal region (Figure 1). Whole-genome single nucleotide polymorphism (SNP) array analysis using the Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA) showed a terminal deletion of 6.4 Mb on chromosome 15q26.2-26.3, which contains disease-causing OMIM genes, NR2F2, IGF1R, MEF2A, ADAMTS17, CERS3, LINS1, ALDH1A3 and CHSY1, and 1.7 Mb duplication on 15q26.2 which does not contain the disease-causing OMIM gene ( Figure 2). No other pathogenic copy number variation or copy number neutral loss of heterozygosity were observed in the SNP array. We diagnosed her with chromosome 15q26-qter deletion syndrome.    FISH was performed using the TelVysion multi-color FISH probe panel No.10. It contains probe for 10p (green), 10q (red), 15q (orange) and 15q22 (blue). The arrow indicates a deletion in the 15q terminal region.
The physical positions correspond to GRCh37/hg19 of the human genome assembly. The arrows indicate the positions of NR2F2 and IGF1R on 15q26.2-26.3, contained in the deletion range.

Discussion
Herein, we describe a 5-month-old girl with a chromosome 15q-terminal deletion syndrome, pre-and postnatal severe growth retardation, microcephaly, CHD, and high fasting GH levels in addition to relatively high IGF-1 levels. Among her 15q-terminal deletions, NR2F2, IGF1R, and MEF2A are genes in which heterozygous deletions cause clinical phenotypes. Among these, heterozygous deletion of NR2F2 cause CHD and heterozygous deletion of IGF1R cause pre-and post-natal severe growth retardation [1,11]. MEF2A has been reported to be associated with autosomal dominant inherited coronary artery disease [12]. Therefore, her current clinical features can be explained by the heterozygous deletion of NR2F2 and IGF1R.
Early diagnostic markers for IGF1R abnormalities have not yet been identified. Most of the patients reported so far have been diagnosed after the age of 2 years due to the short stature of children born SGA. Walenkamp et al. [13] proposed a clinical score to drive molecular investigations of IGF1R abnormalities. This score includes the following 4 criteria: birth length or weight <−1 SD, head circumference at first presentation <−2 SD, height at first presentation <−2.5 SD and serum IGF-1 levels above the mean for age and sex. While our patient was diagnosed in early infancy, she met all of these criteria. However, the initial IGF-1 level was only slightly above the age-average level, making it difficult to suspect IGF1R abnormalities.
It is known that newborns and young infants have low IGF-1 levels due to immaturity of the GH-IGF-1 axis [14]. However, IGF-1 levels in early infancy have not always been reported to be low, reflecting immaturity of the GH-IGF-1 axis. For example, the average IGF-1 level at 3-4 months of age has been reported to be 37.3-93 ng/mL [14]. Since undernourished infants with poor weight gain, such as our case, usually tend to have low IGF-1 levels, the serum IGF-1 levels in the case were determined to be relatively high IGF-1 levels. If IGF-1 levels are low, we also consider abnormalities in GH-IGF-1 signaling pathways such as Laron syndrome, IGF-1 deficiency, STAT5B, ALS, and PAPPA2 deficiencies [15].
Theoretically, in addition to relatively high serum IGF-1 levels, serum GH levels should also be high in IGF1R abnormalities. A summary of IGF1R abnormalities with the described GH levels is presented in Table 2. Previous reports have shown that basal GH levels vary among patients but tend to be higher at younger ages. In the GH stimulation tests, GH peak levels were subnormal to mildly elevated in most cases. All but one of the patients were diagnosed after the age of one year. Typically, GH levels are high at birth, then fall sharply for the first few weeks, and slowly over the next few months reaching pre-pubertal levels around 6 months of age [16]. In particular, SGA newborns had higher serum GH levels at birth than normal newborns in all trimesters, but serum GH levels at 3 days after birth were not significantly different between the 2 groups [17]. Nevertheless, basal GH levels are clearly implicated in the alterations of GH secretion observed in various physiological states (stress, sleep, bleeding, fasting, hypoglycemia, exercise, etc.), and their assessment should be determined in the setting of age, blood glucose levels, and blood sampling procedures. Our patient had significantly higher basal GH levels at 3 months of age, which could be attributed to the testing in early infancy, relatively low blood glucose levels, and venipuncture procedures. High basal GH levels may also be a marker for suspicion of GH-IGF-1 insensitivity disorders in early infancy.
The mechanism of the deletion of the 15q26 region is unclear. The range of IGF1R deletions varies from deletion of the IGF1R exon level to an identifiable size by G-band analysis, with no specific deletion. In previous reports, IGF1R deletions have ranged from 19 kb to 11.2 Mb [8,18]. Besides the deletion of the 15q26.2-26.3 region, a 1.7 Mb duplication was observed in our patient, suggesting that the 15q26-ter deletion may have been caused by an inverted duplication although we have not actually confirmed this. In fact, a 15qterminal deletion by inverted duplication has been reported [19]. The 1.7 Mb duplicated region upstream of the deletion region does not contain any OMIM disease-related genes. Therefore, we consider it unlikely that this duplication is involves in the phenotype.
NR2F2 plays an important role in angiogenesis and heart development by participating in signaling between the endothelial and mesenchymal compartments [20]. The expression of NR2F2 in the developing human fetal heart including the atria, coronary vessels, and aorta has been substantiated, and NR2F2 mutations have been causally linked to isolated congenital heart disease, including atrio ventricular septal defect, tetralogy of Fallot, aortic stenosis, VSD, coarctation of the aorta and hypoplastic left heart syndrome [11]. In this case, loss of function of NR2F2 due to 15q26-ter deletion caused ASD and VSD.
In summary, to our knowledge, this is the first report showing that fasting GH levels are high in early infancy in patients with IGF1R abnormalities. Previous reports may not necessarily have shown high GH levels because the patients with IGF1R abnormalities were diagnosed after the age of 1 year. However, this study has several limitations. Firstly, since blood was drawn at the same time as the venipuncture, a stress-induced GH elevation cannot be ruled out. Secondly, blood glucose levels were not measured at the same time as the GH test except for once; therefore, we do not know the contribution of hypoglycemia to high serum GH levels. Further clinical studies are needed for analyzing more cases. IGF-1, insulin-like growth factor 1; GH, growth hormone; SDS, standard deviation score; NA, data not available; y, years; m, months; M, male; F, female; Peak GH, GH peak was assessed in stimulation test; *, The ranges of IGF-1 levels are those described in the respective manuscripts; **, Patients' IGF-1 SDS values reported in the respective manuscripts.

Conclusions
In addition to relatively high IGF-1 levels, elevated fasting GH levels in early infancy may contribute to the diagnosis of IGF1R abnormalities. Routine testing of specific parameters when growth retardation is detected during pregnancy will definitely enhance the knowledge of the relation to elevated GH and IGF-1 levels. Institutional Review Board Statement: Ethical review and approval was waived for this study due to the following reasons: Our ethics approval is not required for a single case report.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy restrictions.