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 MECP2 duplication syndrome. It can also be used for the detection of deletions or duplications in the human CDKL5, ARX and NTNG1 genes, in order to confirm a potential cause for and clinical diagnosis of CDKL5 deficiency disorder, early infantile epileptic encephalopathy 1 (EIEE1) and atypical Rett syndrome, respectively. This assay is additionally intended for molecular genetic testing of at-risk family members, and 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 each exon of CDKL5, ARX and NTNG1.

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.

¹ Please 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).
² To 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 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 are traditionally subdivided into five distinct clinical subgroups: congenital, early-onset seizure, preserved speech, late regression and forme fruste variants (Hagberg et al. 2002; Hagberg and Skjeldal 1994; Neul et al. 2010). The early onset seizure and congenital variants of RTT are nowadays considered distinct clinical entities: CDKL5 deficiency disorder and FOXG1 syndrome, respectively (Fehr et al. 2013).

RTT is an X-linked dominantly inherited disorder that, in most cases, is caused by mutations of the MECP2 gene encoding methyl-CpG-binding protein 2 (https://www.ncbi.nlm.nih.gov/books/NBK1497/; Amir et al. 1999). Mutations in MECP2 account for 95-97% of the classic RTT cases (Neul et al. 2008; Neul et al. 2010). 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 MECP2 is not required to make the diagnosis of classic RTT. In contrast to classic RTT, mutations in MECP2 have been identified in only 50-70% of atypical RTT cases (Percy et al. 2007). Most cases of RTT are the result of de novo mutations. Approximately 5-10% of the MECP2 mutations are large deletions/duplications (Archer et al. 2006; Hardwick et al. 2007; Pan et al. 2006; Philippe et al. 2006; Zahorakova et al. 2007). Involvement of other genes in atypical RTT has been reported. One report described a patient with atypical RTT who presented with early onset of epileptic seizures (not infantile spasms) and a de novo translocation that disrupted the NTNG1 gene on chromosome 1 (Borg et al. 2005). This balanced translocation will not be detected by MLPA as the NTNG1 copy number is not altered. Deletions and duplications of NTNG1 have not been described so far.

CDKL5 deficiency disorder (previously classified as early onset seizure variant of RTT; also known as early infantile epileptic encephalopathy 2) is a condition characterized by a broad range of clinical symptoms and severity. The primary symptoms include early-onset epilepsy (starting within the first three months of life), generalized hypotonia, psychomotor development disorders, intellectual disability, and cortical vision disorders. CDKL5 deficiency disorder is an X-linked dominantly inherited disorder that is caused by mutations in the CDKL5 gene (Kalscheuer et al. 2003; Scala et al. 2005; Weaving et al. 2004). The prevalence among women is four times higher than in men (Jakimiec et al. 2020), but the course of the disease is usually more severe in male patients. Most cases of CDKL5 deficiency disorder are the result of de novo mutations. It is estimated that ~6.5-10% of the CDKL5 mutations are large deletions or duplications (RettBASE; RettSyndrome.org Variation Database). Mosaicism has been reported for CDKL5 mutations with an overall frequency of 8.8% (Stosser et al. 2018). Large mosaic deletions have also been described (Bartnik et al. 2011; Boutry-Kryza et al. 2014; Mei et al. 2014), but the occurrence rate for mosaic copy number changes has not been determined.

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, speech and motor delay, seizures, hypotonia, and progressive spasticity (https://www.ncbi.nlm.nih.gov/books/NBK1284/).

Early infantile epileptic encephalopathy (EIEE; also known as developmental and epileptic encephalopathy) is a neurological disorder characterized by seizures. The disorder affects male and female 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. EIEE can be caused by mutations in more than 100 different genes. EIEE1 is an X-linked recessive disease that is caused by mutations in the ARX gene. Males with ARX mutations are often more severely affected than females, but female mutation carriers may also be affected (Kato et al. 2004; Wallerstein et al. 2008). Approximately 3% of identified ARX mutations are large deletions and duplications (Shoubridge et al. 2010).

Since there are multiple genes involved in the above-described syndromes and since these genes are covered by two different probemixes, i.e. SALSA MLPA Probemix P015 MECP2 and SALSA MLPA Probemix P189 CDKL5/ARX//FOXG1, Table 1 in the Product Description 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, nine probes are present for genes in close proximity to MECP2. One of these probes detects the VAMP7 gene that is located within the pseudo autosomal region 2 (PAR2). The P015-F2 MECP2 probemix also contains four CDKL5 probes, two ARX probes, and four NTNG1 probes. More probes for the CDKL5, ARX and NTNG1 genes are present in SALSA MLPA Probemix P189 CDKL5/ARX/FOXG1. 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.