With the introduction of a microarray platform with exon coverage, we demonstrate the utility of this technology in detecting intragenic deletions: one in exons 24–27 of the EP300
gene and another in exons 27 and 28 of the CREBBP
gene. These results illustrate that exon-targeted aCGH is a powerful tool for detecting clinically significant intragenic rearrangements that would be otherwise missed by aCGH platforms lacking sufficient exonic coverage or sequencing of the gene of interest. The resolution of the current clinical array used in the clinical laboratory setting is about 300–500
kb, which is not sufficient to detect intragenic changes. This version 8 array was designed with exon coverage to detect deletions and duplications with a resolution between 1000
bp and 300
kb. The continuing improvement of cost-effective high-density arrays enables detection of copy-number changes that are <1000
bp in size in the clinical setting. In fact, the design of the high-density region-specific exon array for specific disease genes such as Duchenne/Becker muscular dystrophy demonstrated the ability to detect deletions and/or duplications involving a single exon.10
An increasing number of new genomic disorders are being described as a result of detection of small intragenic copy-number changes, such as the MEF2C16
genes, from high-density clinical arrays such as the one used in this report. Although other technologies, such as MLPA, can also detect intragenic deletions/duplications, it requires that the clinical phenotype be obvious enough to indicate testing for a specific gene. Whereas, an array with this level of resolution does not have this limitation enhancing its value for diagnosing conditions with variable expressivity or that lack hallmark clinical findings.
A total of 3000 samples were referred and evaluated using this version of the exon array during a period of 6 months. The indications for the study population varied from dysmorphic features, moderate developmental delay, mental retardation, multiple congenital anomalies, congenital heart disease to no indication provided. A total of 25 cases with exon deletions were identified and confirmed by PCR or MLPA and/or sequencing. Among them, three cases were detected with RSTS, although only one case (case 2) had the indication to rule out RSTS. The third case was not reported here due to lack of consent. However, the indications for this case were moderate developmental delay, mental retardation and dysmorphic features. CMA revealed a copy-number loss at band 16p13.3 spanning ~10
kb. This loss deletes exons 11–16 of the CREBBP
gene. This 3.5-year-old child has normal thumbs, but his great toes appear disproportionately large bilaterally as is seen in RSTS. He also has micrognathia, inverted nipples and prominent globes.
RSTS is a complex autosomal dominant disease. Point mutations and small deletions or insertions of the CREBBP
genes3, 18, 19
, as well as deletions and duplications >1000
bp in length to megabases, have been shown to lead to RSTS.3, 20
Stef et al21
also showed that a deletion as small as 3.3
kb involving a few exons of the CREBBP
gene can be detected using a set of seven BACs and 34 cosmids region-specific array covering 2
Mb in the 16p13.3 region. It has been well established that deletions involving the CREBBP
gene are the causative mutation in ~10% of RSTS patient. Traditionally, FISH analysis has been employed to detect these deletions. However, the resolution of FISH analysis is not sufficient for detecting smaller intragenic deletions. With the increased resolution, this exon-targeted array now offers a new opportunity to diagnose patients with RSTS due to copy-number changes in these two genes associated with RSTS in a single assay. It may also prove particularly helpful in diagnosing patients with EP300
copy-number changes as these patients often do not present with the skeletal findings that are a hallmark feature of this syndrome. Therefore, the RSTS provides us an excellent example to illustrate our capability in detecting small CNV to yet a higher level of resolution in the clinical setting, particularly if a specific gene cannot be targeted for testing based on the clinical findings.
Mutations and deletions in EP300
are rare causes of RSTS. Our case is only the seventh case reported to date.3, 4, 11, 12
The true percentage of RSTS caused by EP300
alterations and the full phenotypic picture of EP300
RSTS is therefore unclear. The diagnosis in our case was considered because of the typical RSTS facial dysmorphism. However, due to the absence of broad and angulated thumbs and halluces, microarray testing was initiated first. CREBBP
testing was to be performed as second-tier testing if the microarray was normal. The facial phenotype in our patient resembles the case (patient 2) described by Bartholdi et al4
and Foley et al12
with very distinct arched bushy eyebrows and hirsutism over the lumbar region. summarizes the reported cases of EP300
mutations, including pregnancy history and other physical and developmental features.
Comparison of the features of patients with EP300 mutations
Interestingly, the mother of Bartholdi's second patient presented with preeclampsia, and the mother of Foley's patient had preeclampsia progressing to hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome.4, 12
Our patient's mother also developed preeclampsia; as she went into premature labor, it is possible that there was no chance for HELLP syndrome to develop. This was the third pregnancy of the seven reported cases to have preeclampsia, which we believe is a solid association. However, a literature search revealed no genes associated with EP300
that are known to also be associated with familial preeclampsia or HELLP syndrome. It is possible that as more genetic links are identified for preeclampsia and HELLP syndrome, an association with EP300
may become apparent. Of note, Lachmeijer et al22
performed a genome scan using 293 polymorphic markers in 67 Dutch sib-pair families affected by preeclampsia, eclampsia or HELLP syndrome. Analysis in 38 preeclampsia families showed suggestive evidence for linkage on chromosome 22q at 32.4
c (LOD score of 2.41), which is in close proximity to the EP300
gene and on chromosome 10q at 93.9
c (LOD score of 2.38).
In conclusion, we report on two cases of RSTS due to exonic deletions detected by a clinical microarray analysis and only the seventh case of RSTS due to an EP300 mutation. Both our case and others in the literature with EP300 mutations have a modified RSTS phenotype, and, in particular, we confirm the milder skeletal phenotype. In addition, we propose that preeclampsia during pregnancy may be a new feature for RSTS caused by EP300 mutations. Together, these cases demonstrate the importance of considering EP300 RSTS in patients who have facial features of RSTS but lack broad and angulated thumbs and halluces. As this array can simultaneously evaluate for copy-number changes in both genes, it may be considered as the first line of testing for patients with suspected RSTS. This exon-targeted array may also replace the del/dup assay by MLPA to compliment sequencing analysis for these two genes. Our case demonstrates the utility of microarray for ascertaining atypical cases and further defining the phenotypic spectrum of RSTS.