Here we report on a series of fetuses with CHARGE syndrome using diagnostic criteria adapted to the prenatal situation. All fetuses underwent an extensive necropsy as well as molecular analysis of the CHD7 gene. This study confirms the role of the CHD7 gene in CHARGE syndrome and allows delineation of the antenatal clinical spectrum of CHD7 mutations.
In our series of fetal CHARGE syndrome, our detailed clinicopathological evaluation led to the identification of three constant features: anomalies of the external ear, agenesis/hypoplasia of the semi‐circular canals, and arhinencephaly. Six other features occur frequently (in at least in seven of 10 cases), namely genital anomalies, thymic hypoplasia, ocular coloboma, other CNS anomalies (other than arhinencephaly), choanal atresia/cleft palate, and heart defect.
Bilateral and asymmetric external ear malformations are found in all our cases. As shown in fig 2A, typical features are small, low set, posteriorly rotated ears, with a prominent crux helix, a small or absent lobe, and a triangular shape as described previously.5,15,16
In the literature, internal ear anomalies, ranging from subtle modular deficiencies to Mondini malformation and semi‐circular canal hypoplasia or agenesis, are classically reported in CHARGE syndrome.7,17,18
Semi‐circular hypoplasia/agenesis was reported in up to 100% of cases in the literature.15,18
We confirm these data and emphasise the necessity of detecting temporal bone anomalies though cephalic x ray evaluation for the antenatal diagnosis of CHARGE syndrome (fig 2Ba,b).
Interestingly, among CNS anomalies, arhinencephaly was found in all our cases (fig 2Bc,d). Arhinencephaly has already been reported by Lin et al
in some patients.19
However, its frequency among CHARGE patients has not been evaluated in the literature, aside from the observation of Harvey et al
who found arhinencephaly in 7/7 CHARGE postnatal cases.20
More recently, Chalouhi et al
assessed olfactory deficiency in 14 children with CHARGE syndrome. Half of them were anosmic and the others had olfactory residual function (hyposmic). All nine MRIs showed anomalies of the olfactory tracts and bulbs varying from moderate hypoplasia to complete aplasia, without any relationship between the radiological and functional results.21
Our systematic neuropathological examination clearly shows that arhinencephaly is a constant sign of CHD7
mutation. We propose it should be considered a major sign for prenatal CHARGE diagnosis. Other CNS anomalies included brainstem and cerebellum abnormalities. They were observed in eight of our 10 fetuses, but were reported also in postnatal cohorts.19,20
They are mainly characterised by hypoplasia of the inferior cerebellar vermis and brain stem (cases 1, 5, 7) and severe cerebellar heterotopia (case 8).
The high incidence of heart defects, cleft lip/palate, and brain anomalies in our series when compared to postnatal data can probably be explained by detection by US during pregnancy. It has been shown that CHARGE individuals have increased mortality due to AVSD defects and cerebellar and or brain stem anomalies associated with ventriculomegaly.7,15
Moreover, males have been reported to have an increased mortality compared to females.15,22
This study presents a population of 10 fetuses with a severe phenotype of CHARGE syndrome, with an especially high number of complex cardiopathies, bilateral posterior choanal atresia, tracheo‐oesophageal fistula,22
brain anomalies, and increased representation of males (7/10).
Among other frequent features, genital anomalies deserve special attention: they were found in 7/10 cases. In three male fetuses, they were noticed only at the histological level (Leydig cell rarefaction). To our knowledge these histological anomalies have not been described previously. This may explain the delay in puberty noticed more in males than females that has been reported postnatally.23
In a female fetus, the only genital anomaly found was a hypoplastic ovary. According to the postnatal literature, female genital hypoplasia is rare and micropenis or cryptorchidism are more frequent features observed in males.24
Interestingly, thymus hypoplasia or agenesis was found in seven of out 10 cases. This contrasts with postnatal cases where thymic hypoplasia is rarely reported.5
The association of CHARGE and DiGeorge syndromes reported previously25
suggests a neural crest defect causes the clinical overlap of both syndromes. Whether the CHD7
gene could be responsible for a CHARGE‐DiGeorge association should be tested further, particularly in postnatal patients.
Our study also confirms that growth retardation is usually observed postnatally.26
Indeed, no intrauterine growth retardation was observed in our antenatal series. In addition to the severe feeding problems related to gastro‐oesophageal reflux and brain stem anomalies, the postnatal growth retardation could be explained in part by a pituitary gland dysfunction. Interestingly, CHD7
gene expression is observed within this tissue during the embryonic period.
It is worth stressing that case 9 presented with ectrodactyly (fig 2C), which has not been reported so far in CHARGE syndrome. Four patients presented limb anomalies although minor, such as clinodactyly. Skeletal (including costal and vertebral defects) or renal anomalies were found in four out of 10 cases. Finally, among other features, tracheo‐oesophageal fistulas were noticed twice.
Based on our clinicopathological observations, we consider semicircular canal agenesis as well as arhinencephaly highly predictive diagnostic criteria of CHARGE syndrome. We suggest that they should be added to the two major diagnostic criteria described by Pagon, along with the external ears malformations. Indeed, four of six major criteria of CHARGE (coloboma, choanal atresia and/or cleft lip/palate, heart defect, arhinencephaly, semi‐circular canal agenesis, and external ear anomalies) were necessary and sufficient for the diagnosis of CHARGE in our series. As previously suggested for postnatal cases, minor diagnostic criteria such as facial dysmorphism and renal, digestive, and skeletal anomalies should also be considered for fetal CHARGE syndrome in addition to thymus hypoplasia/agenesis and polyhydramnios. Interestingly, in most patients with three major features and three minor features proposed as CHARGE syndrome,5
the presence of arhinencephaly and semi‐circular canals hypoplasia/agenesis was not evaluated and we recommend brain MRI to assess the diagnosis of CHARGE syndrome in these cases.
Our study contributes to the development of a strategy for the diagnosis of CHARGE syndrome during pregnancy. Indeed, features of CHARGE syndrome detected at routine US such as hydramnios, heart defects, cleft lip/palate, CNS anomalies, and kidney or gastrointestinal anomalies are common. More specific features would help in the diagnosis of CHARGE syndrome. We thus propose that focussed US and/or brain MRI should be performed for the detection of external ear anomalies, choanal atresia, semi‐circular canal agenesis, and arhinencephaly. This strategy will certainly lead to a higher prenatal detection rate of CHARGE syndrome.
Embryonic function of CHD7
The CHD7 protein encompasses several important domains. The chromatin organisation modifier (chromo) domain is a conserved region of around 50 amino acids found in a variety of chromosomal proteins that appear to play a role in the functional organisation of the eukaryotic nucleus. Experimental evidence shows that the chromodomain is involved in binding proteins to histone and possibly RNA. CHD7‐like helicase domains are involved in ATP‐dependent unwinding of DNA or RNA duplexes and histone deacetylation.27,28
Certain CHD proteins have been shown to participate in nucleosome remodeling deacetylase (NuRD) protein complexes which interact with sequence‐specific DNA‐binding factors for targeted repression.29
Presumably, the CHD7 protein plays an important role in chromatin remodelling during early development and allows a level of epigenetic control over target genes expressed in mesenchymal cells derived from the cephalic neural crest.
We analysed the expression pattern of the CHD7
gene during early human development. CHD7
is widely expressed in the undifferentiated neuroepithelium and in mesenchyme of neural crest origin. Towards the end of the first trimester it is expressed in dorsal root ganglia, cranial nerves/ganglia, and auditory, pituitary, and nasal tissues as well as in the neural retina. Absent from the myocardium, bones of mesodermal or neural crest origin, and the genital ridge, CHD7
expression correlates with defects observed in these tissues because of its presence in neural crest cells investing the outflow tract of the heart, and in the hypothalamus and pituitary gland. Endocrine deficiency may occur centrally, in the differentiation of hypothalamic nuclei secreting somatostatin or GnRH, or more peripherally in the differentiation of somatotropic or gonadotropic cells of the anterior pituitary. It could be related to the clinical findings of delayed puberty in the adolescent and adult population with CHARGE syndrome.23
A major testable hypothesis in this context is that CHD7 regulates paired domain‐ or homeobox domain‐containing transcription factor genes important for the development of the pituitary gland and other organ systems, such as Hesx1
, and Titf1/Nkx2a
. Other regulatory transcription factor genes such as Pax2
, with multiple expression sites in many affected organ systems or inductive tissues for these organ anlages, still remain attractive and non‐exclusive CHD7 functional targets.30