Intended purpose
The SALSA MLPA Probemix P189 CDKL5/ARX/FOXG1 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 CDKL5 and FOXG1 genes, in order to confirm a potential cause for and clinical diagnosis of CDKL5 deficiency disorder and FOXG1 syndrome, respectively. It can also be used for the detection of deletions and duplications in the human ARX and NTNG1 genes, in order to confirm a potential cause for and clinical diagnosis of 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 P189 CDKL5/ARX/FOXG1 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 CDKL5, FOXG1, 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.

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 (IVD) use 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
CDKL5 deficiency disorder (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 has several features in common with Rett syndrome and was previously described as the early-onset seizure variant of Rett syndrome (see below). However, as other signs and symptoms of CDKL5 deficiency disorder are distinct from those of Rett syndrome, the disorder is nowadays considered a separate clinical entity (Fehr et al. 2013).

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.

FOXG1 syndrome is a condition characterized by impaired development and structural brain abnormalities. Affected infants are small at birth, and their heads grow more slowly than normal, leading to an unusually small head size (microcephaly) by early childhood. The condition is associated with a particular pattern of brain malformations and affects most aspects of development. Children with the condition typically have severe intellectual disability. FOXG1 syndrome is an autosomal dominant condition that is caused by mutations in the FOXG1 gene (Kortüm et al. 2011; Vegas et al. 2018; Wong et al. 2019). The percentage of FOXG1 syndrome cases explained by large deletions or duplications of FOXG1 varies depending on the phenotype examined, but has been estimated at ~11% (Vegas et al. 2018). FOXG1 syndrome was previously described as the congenital variant of Rett syndrome (see below). However, whereas Rett syndrome is diagnosed almost exclusively in females, FOXG1 syndrome affects both males and females. Because of these differences, FOXG1 syndrome is nowadays considered a distinct disease entity.

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).

Rett syndrome is a neurodevelopmental disorder affecting approximately 1:10,000 live female births. Classic Rett syndrome 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 Rett syndrome are identified as atypical Rett syndrome 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). Most cases of Rett syndrome are caused by de novo mutations in the MECP2 gene (GeneReviews; https://www.ncbi.nlm.nih.gov/books/NBK1497/). However, there is one report that describes a patient with atypical Rett syndrome 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.

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 P189 CDKL5/ARX/FOXG1 and SALSA MLPA Probemix P015 MECP2, 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.

Probemix content
The SALSA MLPA Probemix P189-C2 CDKL5/ARX/FOXG1 contains 52 MLPA probes with amplification products between 130 and 500 nucleotides (nt). This includes 24 probes for the CDKL5 gene, five probes for the ARX gene, 12 probes for the NTNG1 gene and two probes for the FOXG1 gene, covering all exons of these genes. In addition, nine 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.