At the time of evaluation of our clinical data, CHD7
sequencing had been performed in 107 index patients referred to our laboratory because of clinical features suggestive of CHARGE syndrome. Pathogenic mutations were identified in 69 patients (65%), including six patients who had previously tested negative.12
All mutations except two were unique and most mutations had a severe effect on the CHD7
protein, being either nonsense or frameshift mutations (70%).
From both our previous data and a recent report by Arrington et al
, it is known that microdeletions of the chromosome 8q12.1 region, including the CHD7
gene, may also result in CHARGE syndrome.12,15
We excluded the presence of such microdeletions in the patients without CHD7
mutations by MLPA. From these results we conclude that whole gene deletions of the CHD7
gene are not a frequent cause of CHARGE syndrome. Currently, we are extending our MLPA analyses in order to assess for the presence of small intragenic deletions.
In 20 out of 21 families a de novo occurrence of the CHD7 mutation could be proven. In the mother of the sibs with the 5982G>A(W1994X) change, this mutation was present as a somatic mosaicism. It is likely that germline mosaicism exists as well. As a consequence, prenatal diagnosis should be offered to all parents of children with an apparently de novo CHD7 mutation.
Of the 69 CHD7 mutation positive patients, 45 index cases were selected for further clinical study together with two sibs, resulting in a cohort of 47 patients. Due to a short follow‐up period, clinical information was limited in 11 patients, especially regarding hearing, growth, and development. From the data presented in tables 3 and 4 and the detailed clinical description of our patients, it is clear that within the CHD7 mutation positive subset of CHARGE patients an extensive variability in clinical presentation exists, without any obvious genotype‐phenotype correlation. This is best demonstrated in the two sib pairs. In the first sib pair, both twin girls had choanal atresia and a heart defect, but they were discordant for the coloboma and tracheo‐oesophageal fistula. The boys of the other sib pair were discordant for cleft lip/palate, heart defect, tracheo‐oesophageal fistula, coloboma, and hearing loss.
Missense mutations were found in three patients of the clinical study group, one of whom was mildly mentally retarded. The other two had normal levels of intelligence. However, normal intelligence was also present in five patients with a nonsense mutation. Overall clinical comparison of these three patients with a missense mutation with the rest of the study group did not reveal any clear differences. However, it is still possible that less severe mutations (that is, missense mutations) result in a less specific phenotype, not recognised as CHARGE syndrome. Hence, such patients may not be included in this study. On the other hand, patients with a CHD7 deletion may be more severely affected than patients with a CHD7 mutation, especially if multiple adjacent genes are deleted. Further studies are needed to explore this.
In table 5 the frequency of the main features of CHARGE syndrome in our group of CHD7 mutation positive patients is compared with data from the literature.
Table 5The frequencies of characteristic CHARGE findings in a population of CHD7 positive patients compared to the literature
The distribution of features in the clinically diagnosed CHARGE syndrome patients as reviewed by Stromland et al
, Issekutz et al
, and Tellier et al
, is comparable to that in our CHD7
mutation positive patients.3,10,16
This indicates that, within the patient group that fulfils the clinical diagnosis of CHARGE syndrome, there is not a specific subgroup that is more likely to have a CHD7
mutation. None of the clinical features seems to be obligatory for a CHD7
mutation, with the possible exception of vestibular anomalies. Several reports have stressed the high frequency and the high specificity of anomalies of the semicircular canals.1,2,17,18
This was also observed in our cohort of patients. All patients investigated by CT scan or vestibular function tests had either abnormal function or an aplasia of the semicircular canals.
The effect of a CHD7 mutation on a specific organ is variable and does not predict the consequences for other organ systems in which CHD7 is expressed. For instance, a severe heart defect does not exclude normal intelligence (for example, individual 43, tables 3 and 4) and severe mental retardation does not have to be accompanied by severe defects in other organs (for example, individual 8, tables 3 and 4). This results in enormous clinical variability, even within sib pairs.
We carefully tested whether both sets of diagnostic criteria could be applied to our patients (tables 3 and 4).9,11
This was not possible in all cases, since, for example, CT scanning of the temporal bones is required in order to apply the diagnostic criteria proposed by Verloes. For simplification we decided to use the 1998 Blake criteria as listed in table 1 instead of the refined criteria adopted for different age groups.10
Both Blake and Verloes require that at least a coloboma or choanal atresia is present for the diagnosis CHARGE syndrome. Five patients in our study group (individuals 4, 6, 9, 28, and 29 in tables 3 and 4) had neither coloboma nor choanal atresia. Blake et al
argued that the choanae are usually patent when orofacial clefting is present and palatal clefting can be substituted for choanal atresia in the scoring criteria.9
As a consequence, only one patient (individual 28 in tables 3 and 4) failed to fulfil the diagnostic criteria for CHARGE syndrome according to both Blake and Verloes. In 38 patients with features suggestive of CHARGE syndrome, no CHD7
mutation and/or deletion was identified. For 27 of these patients, sufficient clinical data were available to apply the clinical diagnostic criteria. Only two of these 27 CHD7
mutation negative patients fulfilled the diagnostic criteria. In both patients aplasia of the semicircular canals was demonstrated. As a consequence, the positive predictive value of the clinical diagnostic criteria is 36/38 (95%). This is substantiated by the fact that after improvement of the sequencing procedure (see supplemental table I available at http://www.jmedgenet.com/supplemental
), the mutation positive percentage in our first reported cohort reaches 95% (18/19).12
In the context of the previously suggested genetic heterogeneity,19,20,21
this is an interesting observation that needs confirmation.
We would like to stress that CHARGE syndrome remains a clinical diagnosis. Although the high percentage of CHD7 mutations in clinically diagnosed CHARGE syndrome patients indicates that CHD7 is the major gene involved, this diagnosis cannot be rejected based on absence of a CHD7 mutation. On the other hand, based on the clinical criteria alone, one CHD7 positive patient would have been missed in our series.
In conclusion, we confirm that mostly unique CHD7 mutations account for the majority of cases with CHARGE syndrome, with a broad clinical variability and without an obvious genotype‐phenotype correlation. In addition, we provided evidence for germline mosaicism.