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Case Reports
. 2010:2010:854737.
doi: 10.1155/2010/854737. Epub 2010 Jul 27.

Small deletion at the 7q21.2 locus in a CCM family detected by real-time quantitative PCR

Lucia Anna Muscarella  1 , Vito Guarnieri, Michelina Coco, Serena Belli, Paola Parrella, Giuseppe Pulcrano, Domenico Catapano, Vincenzo A D'Angelo, Leopoldo Zelante, Leonardo D'Agruma
Affiliations
Case Reports

Small deletion at the 7q21.2 locus in a CCM family detected by real-time quantitative PCR

Lucia Anna Muscarella et al. J Biomed Biotechnol. 2010.

Abstract

Cerebral cavernous malformations (CCMs) represent a common autosomal dominant disorder that predisposes patients to haemorrhagic strokes and focal neurological signs. About 56% of the hereditary forms of CCMs have been so far associated with mutations in the KRIT1 (Krev Interaction Trapped 1) gene, located at 7q21.2 (CCM1 locus). We described the complete loss of 7q21.2 locus encompassing the KRIT1 gene and 4 flanking genes in a CCM family by using a dense set of 12 microsatellite markers. The complete loss of the maternal copy of KRIT1 gene region was confirmed by Real-Time Quantitative Polymerase Chain Reaction (RT-QPCR) and the same approach was used for expression analysis. Additional RT-QPCR analysis showed the extension of the deletion, for a total of 700 kb, to the adjacent downstream and upstream-located genes, MTERF, AKAP9, CYP51A1, as well as a partial loss of the ANKIB1 gene. Here we report the molecular characterization of an interstitial small genomic deletion of the 7q21.2 region in a CCMs affected family, encompassing the KRIT1 gene. Our findings confirm the loss of function mechanism for the already known CCM1 locus, without any evident involvement of the other deleted genes. Moreover, our investigations highlight the usefulness of the RT-QPCR to the molecular characterization of the breakpoints genomic deletions and to the identification of internal deleted genes involved in the human genetic diseases.

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Figures

Figure 1
Figure 1
Unenhanced CT image of the index case (Case II : 2) showing a large lesion with a hyperattenuating centre surrounded by little spots of increased density (suggestive for calcifications and small areas of haemorrhage) in the left frontal lobe. Large edema coexists. Further histological examination proved this lesion to be a CCM.
Figure 2
Figure 2
(a) Haplotype analysis of microsatellites markers from chromosome 7q21.1. The haplotype shared by the affected individuals (black-filled symbol) is “boxed”. In the affected patients, the symbol (∗) indicates the indefinite alleles, located in the deletion. Since the hemizygosity for the non shared alleles in the affected patients, the analysis, shows “homozygosity” for three markers (D7S2409, D7S1813, D7S1789), thus, the deleted alleles are indicated with the same symbol (∗). (b) Measurements of copy number status (± standard error mean) of genes mapping in the 7q21 chromosomal region determined by RT-QPCR. On the Top: the genomic organization of genes. Genes are shown above the horizontal axis, which also indicates the extension and orientation of each gene. Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed quare (■), and a closed circle (●), respectively. The genes between the two vertical gray dotted lines show a hemizigous deletion detectable by the copy number loss of the associated amplicons.
Figure 3
Figure 3
Copy number status Expression level analysis (± standard error mean) of genes mapping in the 7q21 chromosomal region determined by RT-QPCR was determined by RT-QPCR. Average relative mRNA expression genes level from two affected members of CCM family (II : 1 and II : 3) related to the normal ones as calibrator ( I : 1) to GAPDH gene as reference.
Figure 4
Figure 4
ANKIB1 gene breakpoint determination by RT-QPCR (± standard error mean). Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed square (■), and a closed circle (●), respectively. The ANKIB1 exons on the left of the vertical dotted lines (exon1–10) show the copy number loss of the associated amplicons.
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