In this study, we report the first application of the MLPA technique to screen for COH1 large deletions and duplications. In a group of 14 patients (11 families) with a clinical diagnosis of Cohen syndrome, MLPA allowed us to obtain rapid and high quality results disclosing 11 deleted and 4 duplicated COH1 alleles. The use of MLPA led us to identify all COH1 mutations undetected by conventional screening, suggesting that this technique is an important tool for the molecular characterization of Cohen syndrome.
Our series included 12 patients with true Cohen syndrome and two brothers with an atypical phenotype, lacking microcephaly and truncal obesity. However, the association of retinopathy, neutropenia and facial appearance addressed the clinical diagnosis. Their facial features, although not typical, were not in disagreement with the diagnosis of Cohen syndrome consisting of long face, heavy eyebrows, mildly down-slanting palpebral fissures, prominent root of the nose, normal philtrum and prognatism (). Three patients from two families were children aged less than 5 years. They presented the typical facial features of younger patients, including round face with full lower lip, not excessively short philtrum, slightly downward-slanting eyes with wave-shaped eyelids and less prominent nasal bridge ().10
Copy number changes in COH1
have been previously investigated in patients with Cohen syndrome by qPCR using probes designed on a limited number of exons.10, 16
Only recently, a targeted oligonucleotide array with a median resolution of 200
bp was designed within the gene, which considerably increased the mutation detection rate.11
Using this technique, the authors identified COH1
large deletions in nine patients from seven families, showing that they represent an important cause of Cohen syndrome.11
The present results and our previous study on a group of 18 patients disclosed a total of 21 alleles with point mutations (58%) and 15 with copy number variations (42%), confirming that deletions and duplications account for a significant percentage of COH1
In four patients from three families, MLPA identified a COH1
large deletion sharing the same extent with one previously reported in an isolated Greek Island population, spanning from exons 6 to 16.16
In our patients, the deletion was heterozygous in two families and homozygous in an apparently non-consanguineous family.10
Interestingly, this latter patient displays the same constellation of facial features reported in Greek patients with homozygous deletion including thick hair with low hairline, strabism, lack of nasofrontal angle, short upturned philtrum and prominent maxillary central incisors (patient 5, ).16
Moreover, they show milder microcephaly and more severe visual impairment than the original phenotype described in the Finnish population.4, 16
Our three families with the same deletion encompassing exons 6–16 come from different Italian regions, two in Central Italy and one in Southern Italy. The results obtained by haplotype analysis in these families, in one member of the large Greek consanguineous family previously reported by Bugiani et al16
harboring the 6–16 deletion in homozygous state and in 50 healthy Italian controls, suggest that the recurrent deletion is due to an ancestral founder effect in the Mediterranean area ().
In this study, we also identified two deletions spanning exons 4–16 and 40–43, sharing the same exon coverage with two deletions already reported in the Northern European population.11
Also, in these cases we cannot exclude a founder effect for the deleted alleles. Alternatively, these could be independent mutations favored by the presence of repeated elements located at the break points. Accordingly, RepeatMasker software analysis of the genomic region containing COH1
revealed a higher frequency of LINEs, SINEs and DNA repeat elements in comparison with the average for autosomal sequences.11
In a previous study, it was suggested that the most likely mechanism for genomic rearrangements in the COH1
gene is the non-homologous end joining, leading to non-recurrent deletions.11
Considering our latest results, the non-allelic homologous recombination mechanism cannot be ruled out.
In four patients from three families, MLPA identified three different size duplications spanning exons 4–13, 20–30 and 57–60, respectively. To our knowledge, COH1 intragenic duplications have never been reported in Cohen syndrome.
In one family with two affected sibs (cases 11A/B), we identified a complex rearrangement (p.T3627_H3633delinsI) in cis
with the downstream duplication detected by MLPA. We initially hypothesized that this rearrangement could be located at the break point of the duplication within exon 56. However, sequencing analysis of the long PCR product using a forward primer in intron 59 and a reverse primer in intron 56 indicated that the duplication effectively starts in intron 56, 95
bp after the rearrangement (). This sequence is joined to exon 61 in position g.100953994 (NM_152564) (). As the MLPA probe of exon 61 is located upstream of the junction point () and its signal does not increase, we can suppose that the duplication is not in tandem. The insertion of the duplicated segments within exon 61 creates a premature stop codon after 10 new amino acids of the protein product. Even if detailed mapping of the extent of all the duplications has not yet been undertaken, these rearrangements probably led to a frameshift and a premature truncation of the protein at different levels.
In conclusion, our study confirms that COH1 copy number variations are a frequent cause of Cohen syndrome and consist of intragenic deletions as well as duplications. Therefore, incorporation of detection tools for COH1 copy number variations is mandatory in the molecular diagnosis of Cohen syndrome.