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

Muscarella LA, Guarnieri V, Coco M, Belli S, Parrella P, Pulcrano G, Catapano D, D'Angelo VA, Zelante L, D'Agruma L - J. Biomed. Biotechnol. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza Hospital, 71013 San Giovanni Rotondo (FG), Italy. l.muscarella@operapadrepio.it

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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(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.
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fig2: (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.

Mentions: Haplotype reconstruction of 13 microsatellite markers on chromosome 7q21 showed heterozygosity for markers flanking the CCM1 locus (D7S2409, D7S1813, D7S1789) in the healthy family member (I : 1), whereas all the affected members (I : 2, II : 1, II : 3, III : 1) carried and shared only the maternal haplotype, indicating a hemizygosity of the specific chromosomal region of about 700 kb (Figure 2(a)). The genetic screening of the whole coding regions of the KRIT1 gene gave negative results.


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

Muscarella LA, Guarnieri V, Coco M, Belli S, Parrella P, Pulcrano G, Catapano D, D'Angelo VA, Zelante L, D'Agruma L - J. Biomed. Biotechnol. (2010)

(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.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2926733&req=5

fig2: (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.
Mentions: Haplotype reconstruction of 13 microsatellite markers on chromosome 7q21 showed heterozygosity for markers flanking the CCM1 locus (D7S2409, D7S1813, D7S1789) in the healthy family member (I : 1), whereas all the affected members (I : 2, II : 1, II : 3, III : 1) carried and shared only the maternal haplotype, indicating a hemizygosity of the specific chromosomal region of about 700 kb (Figure 2(a)). The genetic screening of the whole coding regions of the KRIT1 gene gave negative results.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza Hospital, 71013 San Giovanni Rotondo (FG), Italy. l.muscarella@operapadrepio.it

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.

Show MeSH
Related in: MedlinePlus