ABSTRACT

Sotos syndrome belongs to the group of diseases characterised by features such as facial dysmorphism, intellectual disability, hypotonia and overgrowth. Usually, Sotos syndrome is caused by heterozygous mutations in the NSD1 gene at chromosome 5q35 or by large genomic deletions of the same region. Genotype-phenotype correlations have mainly been reported as an association of significant or major abnormalities and presence of 5q35 deletions rather than intragenic deletions or point mutations in NSD1. Congenital hyperinsulinemic hypoglycaemia (CHI) has been described as an uncommon feature in the presentation of Sotos syndrome. Most of the patients with Sotos syndrome and transient CHI were carriers of 5q35 deletions, while persistent CHI has been recently reported in individuals with point mutations or small NSD1 deletions. We report the clinical features and medical treatment in a new-born child with Sotos syndrome and CHI that was present for almost two years. Genetic cause of Sotos syndrome in this case was a novel, large genomic deletion encompassing 24 Online Mendelian Inheritance in Man genes including the entire NSD1 gene and six other potentially morbid genes. Our report describes challenges in diagnosis and management of this rare genetic condition. We propose, that in neonatal diagnostics, the phenotypic spectrum of Sotos syndrome should include CHI as a characteristic feature and molecular genetic testing should be done by whole genome analysis.

Introduction

Sotos syndrome (SOS) [Online Mendelian Inheritance in Man (OMIM) #117550] belongs to a group of congenital overgrowth disorders characterised by facial dysmorphism, brain involvement, prenatal and postnatal overgrowth, cardiac defects, kidney problems, scoliosis and loss of vision and hearing (1, 2).

SOS is caused by haploinsufficiency of the NSD1 gene at 5q35.2-q35.3 coding for nuclear receptor binding SET domain 1 protein. The NSD1 protein functions as a transcriptional regulator of chromatin through the histone methyltransferase activity (1, 3). To date, 623 disease-associated variants in NSD1 have been reported to the Human Gene Mutation Database (HGMD Professional) (https://my.qiagendigitalinsights.com/bbp/view/hgmd/pro/gene.php?gene=NSD1). Most of the variants are missense mutations (n=263), followed by small deletions (n=142) and gross deletions (n=63). Gross deletions may result in removal of a single or several exons or the entire NSD1 gene with adjoining genes. Deletions have been reported to vary from 3.8 kb to 5 Mb, according to HGMD Professional. The majority of the large genomic deletions including NSD1 appear de novo while familial cases with missense mutations in the NSD1 gene have also been reported (1, 4).

Molecular techniques, such as genome-wide genotyping or chromosomal microarray (CMA) and multiplex ligation-dependent probe amplification are usually used for detection of large genomic deletions encompassing NSD1 or the NSD1 intragenic deletions, that can also be confirmed by fluorescence in situ hybridization (FISH).

Congenital hyperinsulinemic hypoglycaemia (CHI) due to inappropriate insulin secretion leading to severe hypoglycemia may be an isolated finding or a feature of the syndrome. CHI has been described as an uncommon feature of SOS, and was initially reported as transient CHI in 1990 (1, 5). Over the last decade, the number of reported cases of SOS with 5q35 deletions and transient CHI became more numerous (6, 7, 8). Most of the cases of SOS with CHI were caused by microdeletions but Sotos patients with transient CHI and point mutations in the NSD1 gene have also been described (6, 9). Thus, Grand et al. (6) presented seven patients, all carriers of NSD1 point mutations, three of whom demonstrated persistent CHI while five of them had atypical features of SOS. These authors concluded that the CHI present in Sotos patients with NSD1 point mutations could not be explained by the deletion of additional genes in the deleted 5q35 region.

A large difference in the frequency of 5q35 microdeletions causing SOS was observed in Japanese (49%) and non-Japanese (6%) patients (10, 11). A partial or whole NSD1 gene deletions were present in ~10% of 30 Brazilian Sotos patients of non-Japanese ancestry (12). In a cohort of Sotos patients from France and the UK, 5q35 the frequency of microdeletions was 18% and 5%, respectively, while intragenic NSD1 mutations responsible for Sotos phenotype were detected in 49% of French and in more than 70% of British patients (13, 14).

In this report, we present clinical features, molecular diagnostics and medical treatment of persistent CHI in a patient with SOS caused by a de novo large genomic deletion encompassing 24 OMIM genes including the entire NSD1 gene.

In this report, we describe the clinical features and medical management of a newborn with SOS complicated by congenital hyperinsulinism, a condition that persisted for nearly two years. We also highlight the diagnostic challenges and therapeutic considerations associated with this rare genetic presentation.

According to national regulations, presentation of this case report did not require approval from an ethics committee. Informed consent for publication was obtained from the patient’s parents.

Case Presentation

A full-term male baby [gestational age of 39 weeks, birth weight 3855 g (+1 standard deviation score, SDS), birth length 53 cm (+1SDS), head circumference 37 cm (+1SDS)], the second child of non-consanguineous Caucasian parents, was born by emergency Cesarean section because of a pathological cardiotocography trace. Antenatal scans showed polyhydramnios, abnormal flow in the umbilical cord and in the arteria cerebri media, as well an abnormal brain morphology. Apgar score was 1-5-10 min: 3-7-8p. Directly after delivery, the patient was found to be hypotonic and hypoglycaemic (P-glucose 0.6 mmol/L) and was admitted to the neonatal intensive care unit. He required intravenous (IV) high concentration glucose infusions with a utilization rate of 13-14 mg/kg/min and, due to tachypnea, he was treated with positive pressure therapy, using continuous positive pressure therapy.

Physical Characteristics

Clinical examination revealed syndromic features, including macrocephaly with prominent forehead, hypertelorism, posteriorly rotated low set ears, short philtrum, flat nasal bridge, and general hypotonia. SOS was suspected.

Systemic Event

At six days of age, he developed repeated seizures, not linked to hypoglycaemia, confirmed with video electroencephalography. Treatment with antiepileptics, phenobarbitone and phenytoin was started. Neuroimaging of brain showed hypo-myelinization, ischemia, a periventricular white matter lesion and reduction of the corpus callosum. A cardiac ultrasound showed a muscular ventricular-septal defect.

Glycemic Event and Treatment

Recurrent hypoglycaemia required continuous glucose infusion and nutritional intake by breastfeeding and nasogastric tube feeding. Repeated diagnostic fast tolerance test was done at the age of 15 days. A critical sample was obtained that revealed plasma-glucose 2.6 mmol/L, C-peptide 0.36 nmol/L, and p-insulin 2.1 mIU/mL. Metabolic investigation for carnitine, methylmalonate, methionine and free amino acids was normal. Plasma beta- hydroxybutyrate was not analyzed and an ammonium level was 67 umol/L, however this was taken at another occasion. The clinical presentation did not resemble hypopituitarism and this diagnosis was excluded because of clinical and laboratory findings.

Diazoxide, as a first-line treatment for CHI, was initiated at a dose 10 mg/kg/day on day 15 with normalisation of p-glucose at day 18. However, due to fluid retention and development of severe pulmonary hypertension with lung oedema and heart failure, diazoxide was discontinued on day 19. The condition of the patient was critical, requiring intubation and respiratory treatment and was regarded as diazoxide “toxicity” affecting the heart. At the intensive care unit, normoglycemia 4-7 mmol/L was observed until patient’s age of 29 days, when he was discharged to the pediatrics care unit.

At day 30, a persistent hypoglycaemia re-occurred and so IV glucose with a utilization rate to 6 mg/kg/min was started. Due to the suspicion of possible diazoxide heart toxicity, octreotide treatment was initiated on day 35, at a dose of 3.5 µg/kg/day and increased by 2 µg/kg/day up to 20 µg/kg/day over 10 days. However, episodes of hypoglycemia persisted. Octreotide treatment was considered ineffective and was discontinued at day 45. On some occasions, diazoxide was carefully re-initiated at low doses of 1 and later 2 mg/kg/day. These doses were well tolerated and therefore were increased to 5 mg/kg/day for maintained normoglycemia. The baby was fed every 2.5 hours and was able to fast for five hours. He maintained normoglycemia at day 46 when glucose infusion was discontinued. Patient’s treatments during the first two months of life are presented in Figure 1.

Follow-up

The patient was discharged from hospital at 12 weeks of age with diazoxide treatment at dose of 5 mg/kg/day. He was mainly fed by nasogastric tube, which was discontinued at six months of age. He was growing at 0SDS for both height and weight, based on Swedish reference and genetic potential was a target height of 0SDS. His head circumference was +3SDS according to the expectations for those with SOS. He was diagnosed with mild mental retardation. At 15 months of age, the patient showed positive progress in motor development. He still required a small dose of diazoxide of 1.5 mg/kg/day and at 18 months diazoxide at the same dose was needed only during infections. At two years of age hypoglycemic episodes completely resolved. Currently he is on a normal diet and can tolerate overnight fast without hypoglycaemia. His heart function was stable at follow-up appointments. The patient was diagnosed with bronchial asthma and treated with conventional inhalation steroid. He has frequently been affected by viral infections complicated by mucus plugs in airways due to hypotonus and has often required short-term hospitalizations. The patient needs a team of specialists for his associated anomalies and development delay.

Materials and Methods

Clinical features, biochemical data, and medical treatments were collected from the patient’s medical records and from personal observations of clinical follow up. Height, weight and head circumference were measured at each visit, and SDS were calculated using current Swedish National references (15, 16).

Genetic Findings

The patient’s DNA analyzed by CMA and revealed a 1349-1354 kb deletion on chromosome 5: arr[GRCh38] 5q35.2q35.3(176,597,879-177,949,621)x1 (Figure 2). The deleted region overlapped 36 HGNC and 24 OMIM genes: GPRIN1, SNCB, UNC5A, HK3, UIMC1, ZNF346, fibroblast growth factor receptor 4 (FGFR4) gene, NSD1, RAB24, MXD3, PRELID1, LMAN2, RGS14, SLC34A1, PFN3, F12, GRK6, DBN1, PDLIM7, DOK3, DDX41, FAM193B, PRR7 and B4GALT7 (Table 1).

The deletion of the NSD1 gene that would result in haploinsufficiency represents the major cause of SOS. Therefore, this loss was interpreted as a pathogenic copy number variant (CNV), causing the syndrome in our patient. The deletion was confirmed by FISH with a locus specific NSD1 probe (Figure 3). The specific signal for NSD1 signal was seen on only one homologous chromosome 5. Subsequent FISH analysis of parental samples did show normal signal pattern with presence of the NSD1 signals on both chromosomes. Thus, we concluded that the deletion in this case appeared to be de novo.

Molecular genetic analyses targeted next generation seuencing with a congenital hyperinsulinism sequencing panel with CNV detection did not identify any pathogenic variants. Minimum NGS coverage ≥20X for all exons and ±10bp of flanking DNA, and ≥10x from 11-20bp of flanking DNA. Average NGS coverage was 165x and fraction of bases covered with NGS was 99.5%. The following genes, ABCC8, GCK, GLUD1, HADH, HNF1A, HNF4A, SLC16A1, and UCP2 were analyzed.

High resolution CMA was performed on peripheral blood collected in EDTA tubes using standard procedure, and DNA was isolated from 200 μL of whole blood using the QiaSymphony (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. DNA was quantified using the Nanodrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

CMA or genome-wide genotyping used for detection of CNVs was performed with the Infinium CytoSNP-850K v1.2 Beadchip (Illumina, San Diego, CA, USA) containing approximately 850,000 single nucleotide polymorphisms markers over the entire genome with an average probe spacing of 1.8kb. Two hundred nanogrammes of ng DNA was hybridized on a beadchip after whole-genome amplification, followed by scanning on the HiScan machine (Illumina). Genotyping results were visualized, normalized and clustered using the Genotyping module of the GenomeStudio software (Illumina) and by BlueFuse Multi software (v.4.4). The cnvPartition 3.2.0 (Illumina) was applied for CNV detection by retrieving Log R Ratio (LRR, the ratio between the observed and the expected probe intensity) and the B allele frequency (BAF). When a CNV is absent, the LRR is around zero, and the BAF is 0, 0.5, or 1 depending on genotypes AA, AB, and BB. Deviations from the expected values indicate copy number alterations. Human genome GRCh38 (NCBI)/hg38 (UCSC) was used for assigning all chromosomal positions. CNVs overlapping with a region of known microdeletion or microduplication syndromes and/or disease-causing genes were classified as pathogenic. The Database of Genomic Variants, the OMIM, and the dbVar Genome Browser and Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources were used to access known microdeletion and microduplication syndromes.

FISH analysis specific for chromosome 5 was performed on metaphase slides according to the manufacturer’s standard protocol (Cytocell Technologies, Cambridge, UK). Probes detecting Cri-du-Chat syndrome on 5p15.2 (CTNND2 in red) and 5p15.31 (UBE2QL1 in green) were used as control probes. The third probe in this mix was a NSD1 specific probe on 5q35 labelled in green. The slides were dehydrated and co‐denatured with the probes at 73 °C for 5 min. Hybridization was done overnight at 37 °C using Hybrite™ (Vysis, Downers Grove, IL, USA). The slides were counterstained with 4’,6‐diamidino‐2‐phenylindole (Vysis). The images were captured by Leica Microscope and analyzed using Cytovision Image Analysis and Capture System (version 7.5) (Leica Biosystems, Maarn, NL).

Discussion and Conclusion

We present a patient with characteristic features of SOS, persistent CHI and a de novo genomic deletion encompassing 24 OMIM genes including the entire NSD1 gene. Among the 24 OMIM genes, seven were classified as morbidity-associated (Table 1). Notably, one of the deleted genes, HK3-hexokinase, a member of the hexokinase family, is involved in the first step of glucose metabolism (17). However, neither sequence variants in HK3 gene or haploinsufficiency have been linked with hyperinsulinism. HK3 sequence variants have been suggested to be associated with ovarian failure and affect the glycolysis important in the development and progression of different neoplasms (18, 19, 20). Another interesting gene is the FGFR4. Sequence variants in FGFR4 gene or haploinsufficiency have been suggested to be associated with diverse phenotypes but not with CHI (12, 21).

A heterozygous mutation in the NSD1 gene (MIM 606681) identified in more than 75% of cases is a common genetic cause of SOS (22). Previously, CHI was reported as an unusual presentation in Sotos patients (5, 23, 24), but in recent years the number of studies reporting CHI in this condition has increased (7, 8). The concurrent presence of NSD1 defects and CHI in SOS has also been reported. Transient neonatal CHI was described in Japanese patients with SOS where 7 of 8 patients harboured a 5q35 microdeletion but only 3 of 8 required diazoxide treatment (7, 8). In a national Japanese survey, CHI was present in about 10% of children with SOS, indicating strong association between these two features (25). Furthermore, CHI was reported in seven patients with SOS caused by point mutations in NSD1 (26). In 3 of 7 patients, CHI persisted for more than one year. These results challenge the previous hypothesis that CHI in SOS is due to the deletion of additional genes in the 5q35 region. Moreover, NSD1 is proposed to play a role in glucose homeostasis. NSD1 was known as a histone methyltransferase and is implicated in the regulation of chromatin and gene expression (6). However, NSD1 is expressed in human pancreatic beta cells, as demonstrated by bulk islet cell analyses and single-cell RNA-sequencing (6, 27, 28). Association between SOS, response to diazoxide treatment and CHI disappearance over time was also described by Kapoor et al. (29), although the exact mechanisms are still not completely understood.

The present case exhibited resolution of his CHI, but it had persisted for almost two years; this is in line with previous publications that reported a similar association between SOS and CHI. The definition of transient hyperinsulinemic hypoglycaemia was poorly defined in earlier studies, and is characterized by spontaneous resolution within a few days but as late as six months of life (29). According to this definition, the transient CHI was prolonged in our case. It is unclear if this is due to the relatively large deletion. The patient required extra feeding and for almost two years was on medication with diazoxide, a ATP-sensitive potassium channel opener, the first-line therapy for CHI (30). It is important to note that our patient responded poorly to diazoxide, which led to heart failure. This meant that when diazoxide was reinitiated, it was at a low dose of less than 5 mg/kg/d because of the risk of heart complications. Nevertheless, the doses were sufficient to avoid hyperinsulinemia and to ensure normoglycaemia. Compared to our patient who required treatment with diazoxide for almost 2 years, previous reports have found diazoxide treatment was required for shorter periods, although this was up to 8 months of age in one report (31) and three children with point mutations in NSD1 were treated over one year (6).

As practice shows, neonatal CHI needs the correct diagnosis and an adequate treatment to avoid neurological consequences. The presented patient with SOS also exhibited a broad spectrum of clinical features, especially in terms of CHI.

The identification of NSD1 abnormalities in most patients with SOS makes a molecular diagnosis possible and helps to confirm a clinical diagnosis of SOS. Despite hypoglycemia being described as a minor feature in SOS, several reports on a genotype-phenotype correlation were published that warrant further research. We propose that, in neonatal diagnostics, the phenotypic spectrum of SOS should include HI as a significant feature.

This case demonstrates that early clinical diagnosis of this rare condition may be challenging and depends on subjective clinical experience and judgement. Experiences and lessons from our management may merit inclusion within medical discourse, and it is hoped this case report will serve as a reference for the diagnosis and treatment of similar patients in the future.