The SALSA MLPA Probemix P015 MECP2 is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative assay 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.

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.

For the full intended purpose, see the product description.

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 three distinct clinical subgroups: congenital, early-onset seizure, and preserved speech (Hagberg et al. 2002; Hagberg and Skjeldal 1994; Neul et al. 2010; Pini et al. 2016). 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, the table below provides an overview of conditions and genes covered by SALSA MLPA Probemix P015-F2 MECP2 and SALSA MLPA Probemix P189-C2 CDKL5/ARX/FOXG1.

SALSA MLPA Probemix P015 MECP2 is CE-marked under the IVDD for in vitro diagnostic (IVD) use in Europe. This assay has also been registered for IVD use in Colombia and Israel.

This assay is for research use only (RUO) in all other territories.

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below have been tested with the current (F2) version of this product and have been shown to produce useful results.

• Coriell NA23599 (f): Heterozygous deletion affecting all probes for MECP2 exon 3-4.
• Coriell NA23635 (f): Heterozygous deletion affecting all probes for MECP2 exon 3 and the probes for MECP2 exon 4 at 356 nt, 229 nt and 346 nt.
• Coriell NA23648 (f): Heterozygous deletion affecting the probes for IRAK1 and the probes for MECP2 exon 4 at 260 nt, 418 nt, 292 nt, 274 nt and 155 nt.
• Coriell NA23654 (f): Heterozygous deletion affecting all probes for MECP2 exon 3 and the probes for MECP2 exon 4 at 356 nt, 229 nt, 346 nt and 155 nt.
• Coriell NA23676 (f): Heterozygous duplication affecting the probes for IRAK1 and MECP2.
• Coriell NA23733 (f): Heterozygous duplication affecting the probes for L1CAM, IRAK1, MECP2 and FLNA.
• Coriell NA23734 (m): Duplication affecting the probes for L1CAM, IRAK1, MECP2 and FLNA.

Various related products

• P075 TCF4-FOXG1: Contains five probes for the FOXG1 gene. The ligation sites of the FOXG1 probes are identical to those in P395 MECF2-FOXG1, with the exception of one of the exon 1 probes.
• P106 X-linked ID: Contains three probes for the ARX gene. The ligation site of the ARX exon 1 probe is identical to the ARX exon 1 probe in P015 MECP2.
• P137 SCN1A: Contains probes for the SCN1A gene. Mutations in SCN1A have been described in two females who fulfil the diagnostic criteria for classic RTT (Henriksen et al. 2018).
• P189 CDKL5/ARX/FOXG1: Contains probes for the CDKL5, NTNG1, ARX and FOXG1 genes.
• P245 Microdeletion Syndromes-1A: Contains three probes for the MECP2 gene. The ligation sites of these probes are identical to probes included in P015 MECP2.
• P395 MEF2C-FOXG1: Contains five probes for the FOXG1 gene. The ligation sites of the FOXG1 probes are identical to those in P075 TCF4-FOXG1, with the exception of one of the exon 1 probes.

General MLPA resources

• MLPA technique overview
• Coffalyser.Net overview

MLPA education

• Getting started with MLPA
• Using Coffalyser.Net to analyse MLPA data
• Help centre with other learning resources

Selected publications using P015 MECP2

• Archer HL et al. (2006). Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients. J Med Genet. 43:451-6.
• Bijlsma EK et al. (2012). Xq28 duplications including MECP2 in five females: Expanding the phenotype to severe mental retardation. Eur J Med Genet. 55:404-13.
• Echenne B et al. (2009). Neurologic aspects of MECP2 gene duplication in male patients. Pediatr Neurol. 41:187-91.
• Fieremans N et al. (2014). De novo MECP2 duplications in two females with intellectual disability and unfavorable complete skewed X-inactivation. Hum Genet. 133:1359-67.
• Hazan F et al. (2021). Clinical Evaluation of Patients with Classical Rett Syndrome and MECP2 Gene Analysis. DEU Trp Derg. 35:87-97.
• Maortua H et al. (2012). CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain. BMC Med Genet. 13:68.
• Sharaf-Eldin WE et al. (2020). Mutation spectrum in the gene encoding methyl-CpG-binding protein 2 in Egyptian patients with Rett syndrome. Meta Gene. 24:100620.
• Vidal S et al. (2019). Characterization of large deletions of the MECP2 gene in Rett syndrome patients by gene dosage analysis. Mol Genet Genomic Med. 7:e793.
• Zhang Q et al. (2019). Genomic mosaicism in the pathogenesis and inheritance of a Rett syndrome cohort. Genet Med. 21:1330-8.

References

• Amir RE et al. (1999). Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet. 23:185-8.
• Archer HL et al. (2006). Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients. J Med Genet. 43:451-6.
• Bartnik M et al. (2011). Early-onset seizures due to mosaic exonic deletions of CDKL5 in a male and two females. Genet Med. 13:447-52.
• Borg I et al. (2005). Disruption of Netrin G1 by a balanced chromosome translocation in a girl with Rett syndrome. Eur J Hum Genet. 13:921-7.
• Boutry-Kryza N et al. (2014). Complex mosaic CDKL5 deletion with two distinct mutant alleles in a 4-year-old girl. Am J Med Genet A. 164A:2025-8.
• Fehr S et al. (2013). The CDKL5 disorder is an independent clinical entity associated with early-onset encephalopathy. Eur J Hum Genet. 21:266-73.
• Hagberg B et al. (1983). A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett's syndrome: report of 35 cases. Ann Neurol. 14:471-9.
• Hagberg B et al. (2002). An update on clinically applicable diagnostic criteria in Rett syndrome. Comments to Rett Syndrome Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology Society Meeting, Baden Baden, Germany, 11 September 2001. Eur J Paediatr Neurol. 6:293-7.
• Hagberg BA et al. (1994). Rett variants: a suggested model for inclusion criteria. Pediatr Neurol. 11:5-11.
• Hardwick SA et al. (2007). Delineation of large deletions of the MECP2 gene in Rett syndrome patients, including a familial case with a male proband. Eur J Hum Genet. 15:1218-29.
• Jakimiec M et al. (2020). CDKL5 Deficiency Disorder-A Complex Epileptic Encephalopathy. Brain Sci. 10:107.
• Kalscheuer VM et al. (2003). Disruption of the serine/threonine kinase 9 gene causes severe X-linked infantile spasms and mental retardation. Am J Hum Genet. 72:1401-11.
• Kato M et al. (2004). Mutations of ARX are associated with striking pleiotropy and consistent genotype-phenotype correlation. Hum Mutat. 23:147-59.
• Mei D et al. (2014). Optimizing the molecular diagnosis of CDKL5 gene-related epileptic encephalopathy in boys. Epilepsia. 55:1748-53.
• Neul JL et al. (2008). Specific mutations in methyl-CpG-binding protein 2 confer different severity in Rett syndrome. Neurology. 70:1313-21.
• Neul JL et al. (2010). Rett syndrome: revised diagnostic criteria and nomenclature. Ann Neurol. 68:944-50.
• Pan H et al. (2006). Large deletions of the MECP2 gene in Chinese patients with classical Rett syndrome. Clin Genet. 70:418-9.
• Percy AK et al. (2007). Rett syndrome: North American database. J Child Neurol. 22:1338-41.
• Philippe C et al. (2006). Spectrum and distribution of MECP2 mutations in 424 Rett syndrome patients: a molecular update. Eur J Med Genet. 49:9-18.
• Pini G et al. (2016). Rett syndrome: a wide clinical and autonomic picture. Orphanet J Rare Dis. 11:132.
• Scala E et al. (2005). Scala E et al. (2005). CDKL5/STK9 is mutated in Rett syndrome variant with infantile spasms. J Med Genet. J Med Genet. 42:103-7.
• Shoubridge C et al. (2010). ARX spectrum disorders: making inroads into the molecular pathology. Hum Mutat. 31:889-900.
• Stosser MB et al. (2018). High frequency of mosaic pathogenic variants in genes causing epilepsy-related neurodevelopmental disorders. Genet Med. 20:403-10.
• Wallerstein R et al. (2008). Expansion of the ARX spectrum. Clin Neurol Neurosurg. 110:631-4.
• Weaving LS et al. (2004). Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation. Am J Hum Genet. 75:1079-93.
• Zahorakova D et al. (2007). Mutation analysis of the MECP2 gene in patients of Slavic origin with Rett syndrome: novel mutations and polymorphisms. J Hum Genet. 52:342-8.