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SALSA MLPA Probemix P015 MECP2

RETT syndrome, classic; RETT syndrome, atypical; MECP2 duplication syndrome; Early infantile epileptic encephalopathy

Region: MECP2 Xq28; CDKL5 Xp22.13; ARX Xp21.3; NTNG1 1p13.3

MLPA | CE IL
Intended purpose
The SALSA MLPA Probemix P015 MECP2 is an in vitro diagnostic (IVD)1 or research use only (RUO) semi-quantitative assay2 for the detection of deletions or duplications in the human MECP2 gene, in order to confirm a potential cause for and clinical diagnosis of classic and atypical Rett syndrome, and of MECP2 duplication syndrome. This assay can also be used for the detection of deletions or duplications in the human NTNG1 and CDKL5 genes, in order to confirm a potential cause for and clinical diagnosis of atypical Rett syndrome, and for the detection of deletions or duplications in the human ARX and CDKL5 genes, in order to confirm a potential cause for and clinical diagnosis of early infantile epileptic encephalopathy (EIEE), and for molecular genetic testing of at-risk family members. This assay is for use with genomic DNA isolated from human peripheral whole blood specimens.

Copy number variations (CNVs) detected with P015 MECP2 should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in the MECP2, CDKL5, ARX and NTNG1 genes are point mutations, none of which will be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis.

Not all exons of the CDKL5, ARX and NTNG1 genes are covered. The SALSA MLPA Probemix P189 CDKL5/ARX/FOXG1 is available for the detection of deletions or duplications in other CDKL5, ARX and NTNG1 exons.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, pre-implantation or prenatal testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

1Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO).
2To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

Clinical background
Rett syndrome (RTT) is a neurodevelopmental disorder affecting approximately 1:10,000-15,000 live female births. Classic RTT is characterized by a period of normal development during the first 6-18 months of life, followed by loss of already gained skills, such as speech and purposeful hand movement. Additional main features are acquired microcephaly, stereotypic hand movements, impaired locomotion and communication dysfunction (Hagberg et al. 1983). Patients, lacking one or more of the major features of RTT, are identified as atypical RTT cases which clustered into five distinct clinical subgroupings; congenital, early-onset seizure, preserved speech, late regression and forme fruste variants (Neul et al. 2010). The prevalence of atypical RTT is estimated at around 1 in 45,000 individuals (predominantly females).

X-linked methyl-CpG-binding protein 2 (MECP2) has been identified as the RTT gene, with mutations in MECP2 found in 95-97% of classic RTT individuals (https://www.ncbi.nlm.nih.gov/books/NBK1497/). Approximately 3-5% of individuals who strictly meet clinical criteria for RTT do not have an identified mutation in MECP2, indicating that a mutation in this gene is not required to make the diagnosis of classic RTT. In contrast to classic RTT, mutations in MECP2 have been identified in only 40% atypical RTT cases. Involvement of other genes in atypical RTT has been reported, such as CDKL5 and NTNG1 (Colak et al. 2011). Approximately 6.5-10% of patients with atypical RTT have large deletions in CDKL5 (RTT database RettBASE). One report described a patient with atypical RTT who presented with early onset of epileptic seizures (not infantile spasms) and a de novo translocation disrupting the NTNG1 gene (Borg et al. 2005). Please note that MLPA will not detect balanced translocations.

FOXG1 syndrome was previously described as a congenital variant of RTT. Both disorders are characterized by impaired development, intellectual disability, and problems with communication and language. However, RTT is diagnosed almost exclusively in females, while FOXG1 syndrome affects both males and females. RTT also involves a period of apparently normal early development that does not occur in FOXG1 syndrome. FOXG1 syndrome is caused by mutations in FOXG1 gene (Kortüm et al. 2011). The SALSA MLPA Probemix P189 CDKL5/ARX/FOXG1 is available for the detection of deletions or duplications in FOXG1 (Table 1).

While loss-of-function mutations in MECP2 result in RTT, gain-of-function mutations are associated with MECP2 duplication syndrome which occurs almost exclusively in males. MECP2 duplication syndrome and RTT share overlapping clinical phenotypes including intellectual disability, motor deficits, epilepsy, hypotonia, and progressive spasticity (https://www.ncbi.nlm.nih.gov/books/NBK1284/).

Early infantile epileptic encephalopathy (EIEE) is a neurological disorder characterized by seizures. The disorder affects newborns, usually within the first three months of life (most often within the first 10 days) in the form of epileptic seizures. Most infants with the disorder show underdevelopment of part or all of the cerebral hemispheres or structural anomalies. The prevalence of EIEE is estimated at around 1 to 1.6 in 100,000 individuals. Mutations in more than 50 different genes are known to cause EIEE, including ARX and CDKL5 (Bahi-Buisson et al. 2010, Kato et al. 2004). Due to the fact that CDKL5 is located on the X chromosome, the prevalence of EIEE among women is four times higher than in men. However, the course is usually more severe among male patients.

Since there are multiple genes involved in the different syndromes and since these genes are covered in multiple probemixes, Table 1 is provided to give an overview.

Probemix content
The SALSA MLPA Probemix P015-F2 MECP2 contains 46 MLPA probes with amplification products between 130 and 467 nucleotides (nt). This includes 17 probes for the MECP2 gene, covering every exon. Furthermore, several probes are present for genes in close proximity to MECP2. One of these probes detects the VAMP7 gene and is located within the pseudo autosomal region 2 (PAR2). The P015-F2 probemix contains four CDKL5 probes, two ARX probes, and four NTNG1 probes. More probes for the CDKL5, ARX and NTNG1 genes are present in the P189 CDKL5/ARX/FOXG1 probemix. In addition, ten reference probes are included that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mrcholland.com.


Order Items

Probemix

Item no.
Description
Technology
Price
P015-025R
SALSA MLPA Probemix P015 MECP2 – 25 rxn
€ 243.00
P015-050R
SALSA MLPA Probemix P015 MECP2 – 50 rxn
€ 486.00
P015-100R
SALSA MLPA Probemix P015 MECP2 – 100 rxn
€ 972.00

Required Reagents (Sold Separately)

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA EK1 reagent kit – 100 rxn – FAM
€ 300.00
EK1-Cy5
SALSA MLPA EK1 reagent kit – 100 rxn – Cy5
€ 300.00
EK5-FAM
SALSA MLPA EK5 reagent kit – 500 rxn – FAM
€ 1380.00
EK5-Cy5
SALSA MLPA EK5 reagent kit – 500 rxn – Cy5
€ 1380.00
EK20-FAM
SALSA MLPA EK20 reagent kit – 2000 rxn – FAM
€ 5295.00

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CE

CE-marked products are for In Vitro Diagnostic (IVD) use only in EU (candidate) member states and members of the European Free Trade Association (EFTA).

IL

IVD-registered in Israel.