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Copy number loss upstream of RAI1 uncovers gene expression regulatory region that may impact Potocki-Lupski syndrome diagnosis.

Alaimo JT, Mullegama SV, Thomas MA, Elsea SH - Mol Cytogenet (2015)

Bottom Line: Understanding the consequence of structural variants such as copy number alterations and their role in gene expression changes is paramount in order to perform a comprehensive analysis of genetic effects on phenotypic variation and disease.Gene expression studies revealed that both the proband and the mother have significantly elevated RAI1 mRNA levels suggesting that the structural variant alters gene expression regulation.Overall, our work demonstrates that the integration of ENCODE data with structural variants of uncertain significance aids in delineating a functional consequence to a genomic aberration and subsequent diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, NAB2015, Houston, TX 77030 USA.

ABSTRACT
The identification of structural variants of uncertain clinical significance is increasing; however, studies delineating the functional consequence of these variants in the pathogenicity of phenotypic features are lacking. Understanding the consequence of structural variants such as copy number alterations and their role in gene expression changes is paramount in order to perform a comprehensive analysis of genetic effects on phenotypic variation and disease. RAI1 is a dosage-sensitive essential neurodevelopmental gene. Copy number loss of RAI1 results in Smith-Magenis syndrome while copy number gain results in Potocki-Lupski syndrome. Here, we present a case of a six year old female with a newly identified maternally inherited copy number loss that lies within the Smith-Magenis syndrome common deletion region, but RAI1 copy number is normal. Integration of the Encyclopedia of DNA Elements (ENCODE) data at the affected region suggests that the deletion disrupts several cis-acting regulatory elements upstream of RAI1, such as multiple repressor sites and an insulator region. Gene expression studies revealed that both the proband and the mother have significantly elevated RAI1 mRNA levels suggesting that the structural variant alters gene expression regulation. The proband and the mother both have some features of Potocki-Lupski syndrome, while the child appears to be more affected with autistic-like features. Overall, our work demonstrates that the integration of ENCODE data with structural variants of uncertain significance aids in delineating a functional consequence to a genomic aberration and subsequent diagnosis.

No MeSH data available.


Related in: MedlinePlus

RT-qPCR expression values of genes within and proximal to 17p11.2 deletion in SMS449 and SMS448. (a) RAI1 gene expression values are significantly reduced in SMS and significantly elevated in PTLS. Both the mother (SMS449) and the proband (SMS448) have significantly elevated RAI1 mRNA levels relative to clinically normal controls, while SMS448 also has significantly elevated levels relative to SMS449. (b) PEMT in both SMS449 and SMS448 have significantly reduced mRNA levels, similar to other individuals with SMS deletions. PEMT levels in individuals with PTLS are significantly elevated (c) FLCN levels are not compromised in either SMS449 and SMS448 despite only having one copy. Results are similar to other SMS deletions. Individuals with PTLS have significantly elevated levels. Data are plotted as means +/-SEM. *p < 0.05 (relative to control) †p < 0.05 (relative to SMS), #p < 0.05 (relative to PTLS), ^p < 0.05 (relative to SMS449)
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Fig3: RT-qPCR expression values of genes within and proximal to 17p11.2 deletion in SMS449 and SMS448. (a) RAI1 gene expression values are significantly reduced in SMS and significantly elevated in PTLS. Both the mother (SMS449) and the proband (SMS448) have significantly elevated RAI1 mRNA levels relative to clinically normal controls, while SMS448 also has significantly elevated levels relative to SMS449. (b) PEMT in both SMS449 and SMS448 have significantly reduced mRNA levels, similar to other individuals with SMS deletions. PEMT levels in individuals with PTLS are significantly elevated (c) FLCN levels are not compromised in either SMS449 and SMS448 despite only having one copy. Results are similar to other SMS deletions. Individuals with PTLS have significantly elevated levels. Data are plotted as means +/-SEM. *p < 0.05 (relative to control) †p < 0.05 (relative to SMS), #p < 0.05 (relative to PTLS), ^p < 0.05 (relative to SMS449)

Mentions: In order to establish RAI1 expression levels for comparison to our patient, we measured RAI1 mRNA levels in lymphoblast cell lines from individuals with SMS and PTLS. As expected, RAI1 mRNA levels were significantly reduced in SMS samples (p = 0.0006) and overexpressed in samples from individuals with PTLS (p < 0.0001) (Fig. 3a). Interestingly, we observed that the proband (SMS448) had a significant increase in RAI1 mRNA levels of ~1.9 fold (p < 0.0001), while maternal (SMS449) expression levels were also significantly elevated to ~1.4 fold relative to controls (p < 0.0001) (Fig. 3a). The paternal RAI1 mRNA level was not significantly different relative to other controls and therefore, was used as a control sample (data not shown). Next, we tested the expression of two other genes in the deletion region to confirm that reduction in gene dosage does alter mRNA levels. First, we tested PEMT in both SMS448 and SMS449 and found significantly reduced mRNA levels relative to normal controls (SMS448; p = 0.0434, SMS449 p = 0.0322) (Fig. 3b). The reduction of PEMT mRNA levels was also observed in individuals with SMS (p = 0.0026) but elevated in individuals with PTLS (p < 0.0001) (Fig. 3b). We also tested the expression level of FLCN and surprisingly found that the mRNA levels were not significantly compromised relative to normal controls in either SMS448 or SMS449 (Fig. 3c). Similarly, for SMS samples, we observed no expression changes in FLCN, despite copy number loss, while a significant increase in mRNA levels was found in PTLS samples (p < 0.0001) (Fig. 3c). Given the FLCN expression data and the lack of clinical findings in both mother and child, BHDS is not likely a medical concern. In summary, expression studies indicate that this particular deletion region increases RAI1 gene expression, which may lead to some features observed in the proband and her mother. However, the mechanism of altered RAI1 gene regulation is unclear.Fig. 3


Copy number loss upstream of RAI1 uncovers gene expression regulatory region that may impact Potocki-Lupski syndrome diagnosis.

Alaimo JT, Mullegama SV, Thomas MA, Elsea SH - Mol Cytogenet (2015)

RT-qPCR expression values of genes within and proximal to 17p11.2 deletion in SMS449 and SMS448. (a) RAI1 gene expression values are significantly reduced in SMS and significantly elevated in PTLS. Both the mother (SMS449) and the proband (SMS448) have significantly elevated RAI1 mRNA levels relative to clinically normal controls, while SMS448 also has significantly elevated levels relative to SMS449. (b) PEMT in both SMS449 and SMS448 have significantly reduced mRNA levels, similar to other individuals with SMS deletions. PEMT levels in individuals with PTLS are significantly elevated (c) FLCN levels are not compromised in either SMS449 and SMS448 despite only having one copy. Results are similar to other SMS deletions. Individuals with PTLS have significantly elevated levels. Data are plotted as means +/-SEM. *p < 0.05 (relative to control) †p < 0.05 (relative to SMS), #p < 0.05 (relative to PTLS), ^p < 0.05 (relative to SMS449)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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Fig3: RT-qPCR expression values of genes within and proximal to 17p11.2 deletion in SMS449 and SMS448. (a) RAI1 gene expression values are significantly reduced in SMS and significantly elevated in PTLS. Both the mother (SMS449) and the proband (SMS448) have significantly elevated RAI1 mRNA levels relative to clinically normal controls, while SMS448 also has significantly elevated levels relative to SMS449. (b) PEMT in both SMS449 and SMS448 have significantly reduced mRNA levels, similar to other individuals with SMS deletions. PEMT levels in individuals with PTLS are significantly elevated (c) FLCN levels are not compromised in either SMS449 and SMS448 despite only having one copy. Results are similar to other SMS deletions. Individuals with PTLS have significantly elevated levels. Data are plotted as means +/-SEM. *p < 0.05 (relative to control) †p < 0.05 (relative to SMS), #p < 0.05 (relative to PTLS), ^p < 0.05 (relative to SMS449)
Mentions: In order to establish RAI1 expression levels for comparison to our patient, we measured RAI1 mRNA levels in lymphoblast cell lines from individuals with SMS and PTLS. As expected, RAI1 mRNA levels were significantly reduced in SMS samples (p = 0.0006) and overexpressed in samples from individuals with PTLS (p < 0.0001) (Fig. 3a). Interestingly, we observed that the proband (SMS448) had a significant increase in RAI1 mRNA levels of ~1.9 fold (p < 0.0001), while maternal (SMS449) expression levels were also significantly elevated to ~1.4 fold relative to controls (p < 0.0001) (Fig. 3a). The paternal RAI1 mRNA level was not significantly different relative to other controls and therefore, was used as a control sample (data not shown). Next, we tested the expression of two other genes in the deletion region to confirm that reduction in gene dosage does alter mRNA levels. First, we tested PEMT in both SMS448 and SMS449 and found significantly reduced mRNA levels relative to normal controls (SMS448; p = 0.0434, SMS449 p = 0.0322) (Fig. 3b). The reduction of PEMT mRNA levels was also observed in individuals with SMS (p = 0.0026) but elevated in individuals with PTLS (p < 0.0001) (Fig. 3b). We also tested the expression level of FLCN and surprisingly found that the mRNA levels were not significantly compromised relative to normal controls in either SMS448 or SMS449 (Fig. 3c). Similarly, for SMS samples, we observed no expression changes in FLCN, despite copy number loss, while a significant increase in mRNA levels was found in PTLS samples (p < 0.0001) (Fig. 3c). Given the FLCN expression data and the lack of clinical findings in both mother and child, BHDS is not likely a medical concern. In summary, expression studies indicate that this particular deletion region increases RAI1 gene expression, which may lead to some features observed in the proband and her mother. However, the mechanism of altered RAI1 gene regulation is unclear.Fig. 3

Bottom Line: Understanding the consequence of structural variants such as copy number alterations and their role in gene expression changes is paramount in order to perform a comprehensive analysis of genetic effects on phenotypic variation and disease.Gene expression studies revealed that both the proband and the mother have significantly elevated RAI1 mRNA levels suggesting that the structural variant alters gene expression regulation.Overall, our work demonstrates that the integration of ENCODE data with structural variants of uncertain significance aids in delineating a functional consequence to a genomic aberration and subsequent diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, NAB2015, Houston, TX 77030 USA.

ABSTRACT
The identification of structural variants of uncertain clinical significance is increasing; however, studies delineating the functional consequence of these variants in the pathogenicity of phenotypic features are lacking. Understanding the consequence of structural variants such as copy number alterations and their role in gene expression changes is paramount in order to perform a comprehensive analysis of genetic effects on phenotypic variation and disease. RAI1 is a dosage-sensitive essential neurodevelopmental gene. Copy number loss of RAI1 results in Smith-Magenis syndrome while copy number gain results in Potocki-Lupski syndrome. Here, we present a case of a six year old female with a newly identified maternally inherited copy number loss that lies within the Smith-Magenis syndrome common deletion region, but RAI1 copy number is normal. Integration of the Encyclopedia of DNA Elements (ENCODE) data at the affected region suggests that the deletion disrupts several cis-acting regulatory elements upstream of RAI1, such as multiple repressor sites and an insulator region. Gene expression studies revealed that both the proband and the mother have significantly elevated RAI1 mRNA levels suggesting that the structural variant alters gene expression regulation. The proband and the mother both have some features of Potocki-Lupski syndrome, while the child appears to be more affected with autistic-like features. Overall, our work demonstrates that the integration of ENCODE data with structural variants of uncertain significance aids in delineating a functional consequence to a genomic aberration and subsequent diagnosis.

No MeSH data available.


Related in: MedlinePlus