The pathogenic mutations, variants, and polymorphisms identified in the current study are listed in Table . Six of them represent DNA changes not previously described in any of the 10 databases consulted [15
]. Two of these new changes were pathogenic, disease-causing mutations found in two patients with early-onset epileptic seizures within the first month of life, and were both de novo
mutations not present in their parents.
CDKL5 mutation/variant identified in this study
The first of these two new mutations was a pathogenic frameshift mutation in exon 8 (c.509_510insGT; p.Glu170GlyfsX36) producing a truncating protein in the catalytic domain that contains only 206 amino-acids on the 1,030 that has CDKL5 protein. The patient was the first child of healthy parents. She was born in a private clinic, and we were unable to obtain all the details related to the delivery, but established that resuscitation had not been required and the infant’s birth weight was 3,450
g. The infant started to have seizures at 4
weeks of life, with facial flushing, vomiting and crying. The family contacted us when the patient was 11
years old with a clinical diagnosis of RTT, with severe mental and psychomotor retardation, poor social contact, axial hypotonia, spastic tetraparesis and epilepsy refractory to numerous treatments.
The second new mutation was a complete deletion of exon 10 (p.745-?_825
?del) detected by MLPA (Figure A) and confirmed by cDNA analysis (Figure B). Although this was an in-frame deletion, we considered it likely to be pathogenic for several reasons: 1) it was a de novo
mutation; 2) all exon deletions from exons 1 to 15 in the genomic databases consulted [16
] were pathogenic; 3) it was located within the catalytic domain; and 4) the clinical description of the patient was consistent with that of others with CDKL5
deletions. This patient was referred to the Clinical Genetics Unit at 4
years old because of delayed psychomotor development, gait abnormalities, growth retardation and severe mental retardation. She was the first child of healthy parents, born by normal delivery after an uncomplicated pregnancy. She had her first epileptic seizures at 4
weeks old (2–3 times a day) and these seizures were refractory to treatment. Follow-up until the most recent visit, at age 6
years, indicated severe and progressive deterioration of her psychomotor development.
Complete deletion of exon 10 detected by MLPA (A) and confirmed by cDNA analysis (B).
Four other new DNA variants were identified, including c.1455_1460delGGCCAA (loss of lysine and alanine amino acids) in exon 12. This patient was referred to us at 3
months old by a paediatric neurologist because of developmental delay, absence of speech, seizures, and difficulty in walking (possible ataxia), though her initial psychomotor development was normal. She was the second child of healthy parents, and had a healthy 13-year-old sister. She had her first febrile seizure at the age of 11
months, and her mother reported that her development “stopped” after this episode, including with respect to her speech (possible regression). We initially classified this DNA variant as non-pathogenic because her asymptomatic mother had the same variant, and both the mother and infant showed the same cDNA and random X chromosome inactivation. However, this variant was not present in the 10 genomic databases consulted, or in the 100 normal control samples, and we therefore reclassified it as an unknown variation.
The second DNA variant was c.2389
A (p. Asp797Asn) in exon 17. This was inherited from the father and was therefore not a pathogenic mutation. Six other females in the healthy control group also showed this change, indicating that it represents a newly-described polymorphism in Spain.
The remaining two novel DNA changes had no clinical significance: c.−426
G and c.403
G. The first was also found in five healthy female controls, and thus represented another novel polymorphism for Spain. In silico
studies of the second change using the ESE Finder program [25
] indicated that it was non-pathogenic. Furthermore, a similar change (c.403
A) in the 1,000 genomes database [24
] was classified as a single nucleotide polymorphism (SNP) variant. No parental DNA was available for either of these cases.
Finally, we detected three previously reported variants. The variant c.2995
A (p.Val999Met), first described by Nectoux et al.
] as likely non-pathogenic and later classified as a polymorphism [27
] and SNP (rs35693326 [18
]), was found in a patient whose asymptomatic mother also had this variant. This variant was also identified among the control group, and we therefore concluded that it represented a gene polymorphism. c.−391
T first reported by Evans et al.
] was found in two patients and four controls. Finally, the haplotype c.145
G; c. 3003
G and c. 3084
A, first described by Tao J et al.
], was found in another two patients.