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Sotos syndrome (SS) or cerebral gigantism (OMIM *117550) is characterised by excessive growth, advanced bone age, typical facial gestalt, and developmental delay.1,2 In infancy growth is rapid, but settles down above the >97th centile in early childhood3,4 and tends to follow this during childhood. The adult height remains close to normal.4 The hands and feet are large. The facial gestalt is very characteristic during childhood with macrocephaly (>97th centile), frontal bossing, prognathism, hypertelorism, and antimongoloid slant of the palpebral fissures. With increasing age, the face gradually lengthens, the jaw becomes more prominent, and macrocephaly is no longer pronounced.5,6 Neurological features are variable and include hypotonia and delay in motor and language development, with a tendency for improvement with age.1,2,5–9 Familial SS is rare. Only 17 families have been reported, most of which show an autosomal dominant mode of inheritance.5,6,10–19
Recent advances have shown a molecular genetic basis for sporadic SS. Initially, two SS patients carrying de novo balanced translocations suggested 5q35 as a possible locus for the SS gene.20,21 Subsequently, the NSD1 gene was isolated from the 5q35 breakpoint of the patient with t(5;8)(q35;q24.1) by positional cloning.22,23NSD1 has an open reading frame of 8088 bp and consists of at least 23 exons (GenBank accession No AF395588). NSD1 is expressed in the fetal/adult brain, kidney, skeletal muscle, spleen, and the thymus, and faintly in the lung.22 The gene encodes 2696 amino acids with SET (su(var)3–9, enhancer-of-zeste, trithorax), PHD (plant homeodomain protein) finger, and PWWP (proline-tryptophan-tryptophan-proline) domains, all of which are possibly related to chromatin regulation, and possibly interact with nuclear receptors (Nrs).22 Among 42 sporadic SS patients examined by direct sequencing, altogether four de novo point mutations predicting truncation of the protein and 20 submicroscopic deletions involving the whole of NSD1 were identified. Two-thirds of SS cases showed NSD1 mutations, so haploinsufficiency of NSD1 is suggested to be the major molecular defect in SS.23 The role of NSD1 in the pathogenesis of SS could be mediated through its action as a corepressor of genes that promote growth.23,25
In this study, we describe a Finnish family with autosomal dominant segregation of classical SS, molecular genetic findings, and FISH analysis of an obvious candidate gene, NSD1.
Patient 1 (son)
The proband is the first child of his parents, but the mother has a healthy daughter from a previous marriage. The pregnancy was complicated by diet treated gestational diabetes. The baby was born prematurely by vaginal delivery, large for gestational age (table 1). Apgar score was 9. The neonatal period was complicated by hypotonia, feeding difficulties, and transient hypoglycaemia, requiring nasogastric tube feeding for six weeks. X ray showed an abnormally elongated shape of the skull and abnormally positioned orbits. EEG showed non-specific abnormalities. Abdominal ultrasound examination and karyotype of cultured peripheral blood lymphocytes were normal.
Sotos syndrome (SS) is characterised by excessive growth, characteristic facial gestalt, advanced bone age, and early developmental delay with a non-progressive or receding course with age.
Recently, deletions and four different point mutations of the NSD1 gene were found to be responsible for Sotos syndrome (SS) in 24 out of 42 Japanese patients, all of whom were sporadic. Although >98% of SS patients have a negative family history, a few families with confirmed parent to offspring inheritance have been described.
Here we report the first NSD1 gene mutation segregating in a Finnish family with two members with classical SS. Both a male infant and his father had a novel 896delC mutation resulting in a frameshift and premature stop codon at nucleotides 955–957 of the NSD1 gene.
These findings confirm that sporadic and familial SS comprise a single entity and both can be caused by mutations in the NSD1 gene.
At the age of 4 months (not corrected for prematurity), the baby was remarkably hypotonic and drowsy. He was tall (+2.1 SD) and macrocephalic (+3 SD) and his height adjusted weight was +10% (8460 g). His bone age was between 9 and 12 months. His facial characteristics included slight hypertelorism and inner epicanthic folds (fig 1). The forehead was slightly prominent with bizygomatic narrowing and frontoparietal baldness. The palpebral fissures were straight and narrow. The jaw was small and the palate was high. He had slightly oedematous hands and feet, and his toenails were deep set. His hands and feet were fairly large, but within normal limits, and there were bilateral simian creases. The truncal skin was loose. The deep tendon reflexes were slightly brisk. His psychomotor development was delayed in terms of lack of reciprocal smile, poor fixation of eyes, and marked hypotonia. Ophthalmological examination showed hyperopia of +3 D and the papillae were pale. The electroretinogram was normal, but visual evoked potentials to flash stimuli showed extended latency with normal potentials. The magnetic resonance imaging (MRI) of the brain showed dilated lateral ventricles and wide frontal cortical spaces, scarcity of periventricular white matter on the trigonum, and a thin corpus callosum. Laboratory tests showed normal blood haemoglobin level, serum creatinine kinase, alanine aminotransferase, whole blood pyruvate, and plasma lactate, and slightly raised ionised calcium in plasma. Based on these data, a clinical diagnosis of Sotos syndrome was made.
His growth has continued to be rapid and his psychomotor development has progressed well, in terms of sitting at the age of 9 months, crawling and getting up to standing position at the age of 14 months, and walking with support soon after. At 16 months, he spoke no words but was babbling. Hypotonia was milder, but poor motor skills of the oral and throat region affected both speech and consuming liquid nutriments, and he drooled abundantly. At present, he receives physiotherapeutic and logopedic support. Owing to hyperopia and strabismus, a spectacle correction of +4.0 D for both eyes was recommended at the age of 13 months.
Patient 2 (father)
The father is a 29 year old male, born as the second child of the family. At birth, he was large for gestational age despite maternal pre-eclampsia. His Apgar scores were 7. Neonatally, he showed hypoglycaemia, hypotonia, a hoarse cry, and poor sucking requiring nasogastric tube feeding for 4 weeks. A systolic heart murmur was heard, which disappeared before 6 months of age.
His growth was rapid and he was macrocephalic (table 1). His motor development was delayed, and at 5 years he was reinvestigated because of hypotonia and delayed speech. At 6 months of age the EEG was normal, but brain ultrasound examination showed mildly dilated lateral ventricles. He had brisk deep tendon reflexes in his lower extremities associated with clonus of the right ankle. The visual acuity was normal, but eye examination showed a few pigmentations below the right papilla. At 5 years, hand x ray showed slightly advanced bone age with dysharmonic maturation, phalangeal age being more advanced than carpal bone age. His phenotype was consistent with Sotos syndrome. Despite delayed early developmental milestones, he attended regular primary and senior high schools, and received occupational education in both commercial and nursing colleges. Presently, he is working at a day care centre for children.
When re-examined together with his son, he had close to normal stature with macrocephaly and long extremities (table 1). He had long facies and a narrow midface giving an impression of hypertelorism, and his palpebral fissures slanted downwards (fig 1). His forehead was slightly prominent with normal hair in the frontoparietal regions. The mandible was prognathic with a prominent pointed chin. The nose and cheeks were erythematous. The palate was high, the teeth were maloccluded, and four of the permanent teeth were lost. His hands and feet were large.
MOLECULAR STUDIES OF THE NSD1 GENE
For FISH, the PAC clone RP1–118m12 (ResGen Invitrogen Corporation, AL) was labelled with FITC-dUTP (DuPont, Boston, MA) and hybridised to peripheral blood metaphase spreads using standard methods. The results were analysed using a Zeiss Axioplan 2 (Carl Zeiss Jena GmbH, Jena, Germany) epifluorescence microscope equipped with a cooled CCD camera (Sensys, Photometrics Ltd, Tucson, AZ) interfaced to the ISIS imaging system (MetaSystems, Altslussheim, Germany).
Green signals were detected on both chromosome 5q35 regions. No deletion was found by FISH analysis (data not shown).
After obtaining informed consent, 5 ml of EDTA blood were drawn from the two patients with SS and the healthy mother of the family. Genomic DNA was extracted from peripheral blood leucocytes following standard methods. Altogether 22 NSD1 exons (exons 2–23) covering the coding region of NSD122 were amplified by PCR. The primer sequences are available on request.23 PCR was cycled 35 times at 95°C for 30 seconds, 50°C for 30 seconds, 72°C for one minute in a volume of 50 μl, containing 1 × PCR buffer with 1.5 mmol/l MgCl2, 0.2 mmol/l each dNTP, 1 μmol/l each primer, and 2.5 U TaqGold polymerase (PE Applied Biosystems). PCR products were purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, CA) and sequenced individually on both strands with BigDye Terminator chemistry by a standard protocol (PE Applied Biosystems) as described previously.24
Genomic sequence analysis of NSD1 showed a heterozygous deletion of a C at position 896 (896delC) in exon 2 in both affected subjects (fig 2). This change results in a frameshift and premature stop codon at nucleotides 955–957, resulting in truncation of 88% of the predicted polypeptide. The mutant allele was not found in the unaffected mother nor in 94 unrelated subjects from the Finnish population (data not shown).
We report the first mutation in the NSD1 gene segregating in a family with SS, confirming that the familial form of this syndrome may be caused by mutations in the NSD1 gene, haploinsufficiency of which has recently been shown to be the major cause of sporadic Sotos syndrome.23
Previous clinical studies have shown that mental retardation is variable and not a constant feature of SS,2 and especially in familial cases the early developmental delay tends to recede with age. Interestingly, the milder clinical picture in this family was associated with an early truncating point mutation in the NSD1 gene, whereas the majority of sporadic patients have a deletion of the whole NSD1 gene along with several other genes residing on the 2.2 Mb segment of deleted DNA. The early developmental course of SS between patients with point mutations and larger deletion may thus be overlapping, although more severe and constant mental retardation may be associated with larger deletions. This is the first report providing preliminary evidence for the phenotype-genotype correlation in Sotos syndrome.
The spectrum of other clinical features appears to be similar in our two patients with a single nucleotide deletion and classical SS patients. In addition to rapid pre- and postnatal growth with a slightly advanced bone age, typical development of the facial gestalt, and early developmental delay, our patients also presented with several other occasional or transient features associated with SS, such as ocular26,27 and neuroradiological findings,7 suggesting a major pleiotropic role of the NSD1 gene in SS.
Familial Sotos syndrome is rare and many of these diagnoses have even been questioned afterwards because of lack of sufficient data.5,6 For this reason, figures for the incidence of sporadic SS is unknown. In the past, the absence of biochemical and molecular markers has made it difficult to distinguish between Sotos and other overgrowth conditions with varying degree of clumsiness and developmental delay.5 Particularly in adulthood, the diagnosis of SS may be challenging because the increased height and macrocephaly are not as pronounced as in childhood and the criterion of advanced bone age cannot be applied. It is not unusual that the diagnosis of SS is made in an adult via an affected child. Nowadays, confirmation of the SS diagnosis by genetic testing is available for approximately 50–70% of patients, allowing more precise genetic counselling and more accurate figures of incidence for both familial and sporadic SS.
Previously, over-representation of sporadic patients with SS has been explained by high mutation rate and reduced reproduction.5 Mental retardation, if present, is an independent factor affecting reproductive fitness. The early developmental delay, or slow normal development, observed in the father of our family faded during childhood enabling normal adult life and transmission of the mutated NSD1 to the next generation. According to our data, the 896delC mutation of the NSD1 gene does not have a major effect on fertility.
We thank Dr T C Cole and Dr Irma Järvelä for helpful discussions and Ms Kazumi Miyazaki, Naoko Takaki, Naoko Yanai, and Yasuko Noguchi for their technical assistance. This work was supported in part by a grant, CREST, from the Japan Science and Technology Corporation (JST) (NK, NM), Helsinki University Hospital Funds (PH), and the Foundation for Pediatric Research in Finland (PH). The authors also thank the family for their kind cooperation.