Jytte Bieber Nielsen

Early onset seizures and Rett-like features associated with mutations in CDKL5

Mutations in the CDKL5 gene (also known as STK9) have recently been shown to cause early onset epilepsy and severe mental retardation (ISSX or West syndrome). Patients with CDKL5 mutations sometimes also show features similar to those seen in Rett Syndrome (RTT). We have screened the CDKL5 gene in 94 patients with RTT or a RTT-like phenotype who had tested negative for MECP2 mutations (13 classical RTT female subjects, 25 atypical RTT female subjects, 40 RTT-like female and 16 RTT-like male subjects; 33 of the patients had early onset seizures). Novel pathogenic CDKL5 mutations were identified in three girls, two of whom had initially been diagnosed with the early onset seizure variant of RTT and the other with early onset seizures and some features of RTT. In addition, the 33 patients with early seizures were screened for the most common mutations in the ARX gene but none were found. Combining our three new cases with the previously published cases, 13/14 patients with CDKL5 mutations presented with seizures before the age of 3 months.

DXA measurements in rett syndrome reveal small bones with low bone mass

Low bone mass is reported in growth‐retarded patients harboring mutations in the X‐linked methyl‐CpG‐binding protein 2 (MECP2) gene causing Rett syndrome (RTT). We present the first study addressing both bone mineral density (BMD) and bone size in RTT. Our object was to determine whether patients with RTT do have low BMD when correcting for smaller bones by examination with dual‐energy X‐ray absorptiometry (DXA). We compared areal BMD (aBMDspine and aBMDtotal hip) and volumetric bone mineral apparent density (vBMADspine and vBMADneck) in 61 patients and 122 matched healthy controls. Further, spine and hip aBMD and vBMAD of patients were associated with clinical risk factors of low BMD, low‐energy fractures, MECP2 mutation groups, and X chromosome inactivation (XCI). Patients with RTT had reduced bone size on the order of 10% and showed lower values of spine and hip aBMD and vBMAD (p < .001) adjusted for age, pubertal status, and body mass index (BMI). aBMDspine, vBMADspine, and aBMDtotal hip were associated with low‐energy fractures (p < .05). Walking was significantly associated to aBMDtotal hip and vBMADneck adjusted for age and body mass index (BMI). Further, vBMADneck was significantly associated to a diagnosis of epilepsy, antiepileptic treatment, and MECP2 mutation group, but none of the associations with vBMADneck remained clinically significant in a multiple adjusted model including age and BMI. Neither aBMDspine, vBMADspine, nor aBMDtotal hip were significantly associated with epilepsy, antiepileptic treatment, MECP2 mutation group, XCI, or vitamin D status. Low bone mass and small bones are evident in RTT, indicating an apparent low‐bone‐formation phenotype.

Mutations found within exon 1 of MECP2 in Danish patients with Rett syndrome

To the Editor: Rett syndrome (RTT; MIM 312750) is a neurodevelopmental disorder almost exclusively diagnosed in females. From the age of 6–18 months, affected persons suffer a gradual reduction of acquired purposeful hand use and speech, accompanied by delay in head growth and temporary loss of contact, manual and gait dyspraxia, seizures and respiratory dysfunction. Repetitive, stereotyped hand movements appear and are a hallmark of the disease (1). In 1999, mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2; MIM 300005) were first reported in RTT patients (2). MECP2 consists of four exons (3). Originally, MECP2 was thought to produce only one transcript including all four exons with a translation start site in exon 2 (MeCP2_e2). Two recent studies have identified an alternatively spliced transcript consisting of exons 1, 3 and 4 leading to the new MeCP2_e1 isoform (4, 5). A classic RTT patient with a mutation within exon 1 was also described (4). The MeCP2_e1 protein appears to be the predominant isoform in the human brain (4, 5). About 85% of the classic RTT patients harbor a de novomutation in the coding region of exon 3 or 4 of MECP2 or large genomic rearrangements (6). In contrast, no mutations specific to exon 2 have been identified. Since exon 1 has been considered to be a non-coding exon, it has not been incorporated in the normal DNA screening for RTT. Evans et al. (7) sequenced exon 1 from 97 RTT patients, who had previously been tested for MECP2 mutations in exons 2–4. Their results were negative and they concluded that mutations in this exon are not common. The findings of the new MECP2 transcript led us to look for mutations in exon 1 in the Danish patient group. We identified two mutations in a group of 10 Danish RTT patients by direct sequencing of exon 1 and the promoter region of MECP2. The 10 patients had previously been tested negative for mutations, first by direct sequencing of MECP2 exons 2, 3 and 4 and second by multiplex ligation-dependent probe amplification technique of all four exons. Patient 1 was a female, born in 1977, diagnosed with classic RTT in 1987 and died in 2002. We identified a novel duplication of 5 bp (CGCCG) in the open-reading frame of exon 1. The mutation, c.23_27dupCGCCG, causes a frameshift, introducing a stop codon downstream after 44 missense amino acids (p.Ser10ArgfsX45) (Fig. 1b). The mutation was not detected in DNA from the parents. Patient 2 is a female, born in 1968 and diagnosed with classic RTT in 1987. Sequencing analysis showed an 11 bp deletion, c.47_57del. The mutation creates a frameshift and a premature stopcodon (p.Gly16GlufsX22) (Fig. 1c). Neither of the parents harbor the mutation. Thus far, only one mutation within exon 1 has been reported (4). It is noteworthy that this mutation is identical with the mutation we found in patient 2. Two additional classic RTT patients have been reported with deletions of exon 1, or involving both exons 1 and 2, respectively (4, 8). Recently, we have found a deletion covering exons 1 and 2 in a third classic RTT patient (9). As exon 2 mutations have never been reported to solely cause RTT, this strengthens the hypothesis that loss of MeCP2_e1 is sufficient to cause classic RTT and an intact MeCP2_e2 is not capable of compensating for this loss. Of 69 patients with classic RTT in Denmark, 60 harbor mutations in exon 3 or 4, and an additional four patients have been identified with large deletions that cover both exons 3 and 4. With our new findings of two exon 1 mutations, the overall mutation detection rate in this group is 96% (66/69). 9% of the mutations in our subgroup of classic patients would not have been found by conventional sequencing of exons 2, 3 and 4. Mutations in exon 1 may be more common than previously reported, but may be restricted Clin Genet 2005: 67: 532–533 Copyright # Blackwell Munksgaard 2005 Printed in Singapore. All rights reserved CLINICALGENETICS doi: 10.1111/j.1399-0004.2005.00444.x

Patients With Rett Syndrome Sustain Low-Energy Fractures

We present the first case-control study addressing both fracture occurrence and fracture mechanisms in Rett syndrome (RTT). Two previous studies have shown increased fracture risk in RTT. This was also our hypothesis regarding the Danish RTT population. Therefore, we investigated risk factors associated with low-energy trauma and the association to methyl-CpG-binding protein 2 (MECP2) mutations. A total of 61 female patients with RTT and 122 healthy controls matched according to age and pubertal/menopause status were examined by questionnaires, bone biochemical markers in blood, and clinical and x-ray evaluations. National register search on fracture diagnoses was done to obtain complete fracture histories. Our results showed that patients with RTT sustained significantly more low-energy fractures from early age compared with controls, even though overall fracture occurrence apparently was not increased. Low-energy fractures were significantly associated with less mobility and lack of ambulation. Associations with MECP2 mutations or epilepsy were not demonstrated, contrary to previous findings. Blood biochemistry indicated a possible need for D vitamin supplementation in RTT. Our study casts light on fracture occurrence in RTT and points to a need for future research in bone development and fracture risk to establish directions for improved prevention and treatment of low-energy fractures in RTT.

A 77-year-old woman and a preserved speech variant among the Danish Rett patients with mutations in MECP2

Two cases with Rett syndrome (RTT) are presented. One of these is a 77-year-old woman. The occurrence of elderly women with diagnosed RTT is sparse; this may be due to the fact that the clinical traits of RTT often are atypical in adult women and the information about early childhood limited. The finding of mutations in the MECP2 gene in many patients with RTT has provided us with a tool for verification of suspected cases. The patient presented here was clinically diagnosed with RTT at the age of 66 years and now the presence of one of the common missense mutations in MECP2 has been demonstrated. Additionally, skewed X-chromosome inactivation was found. It is likely that this woman is the one of the oldest living patients with RTT. The other case is a 30-year-old woman with preserved speech and the MECP2 missense mutation R133C. Possibly this mutation gives rise to a relatively mild phenotype. A survey of the 44 mutations in the Danish patient group is given.

Two new Rett syndrome families and review of the literature: expanding the knowledge of MECP2 frameshift mutations

BackgroundRett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder, which is usually caused by de novo mutations in the MECP2 gene. More than 70% of the disease causing MECP2 mutations are eight recurrent C to T transitions, which almost exclusively arise on the paternally derived X chromosome. About 10% of the RTT cases have a C-terminal frameshift deletion in MECP2. Only few RTT families with a segregating MECP2 mutation, which affects female carriers with a phenotype of mental retardation or RTT, have been reported in the literature. In this study we describe two new RTT families with three and four individuals, respectively, and review the literature comparing the type of mutations and phenotypes observed in RTT families with those observed in sporadic cases. Based on these observations we also investigated origin of mutation segregation to further improve genetic counselling.MethodsMECP2 mutations were identified by direct sequencing. XCI studies were performed using the X-linked androgen receptor (AR) locus. The parental origin of de novo MECP2 frameshift mutations was investigated using intronic SNPs.ResultsIn both families a C-terminal frameshift mutation segregates. Clinical features of the mutation carriers vary from classical RTT to mild mental retardation. XCI profiles of the female carriers correlate to their respective geno-/phenotypes. The majority of the de novo frameshift mutations occur on the paternally derived X chromosome (7/9 cases), without a paternal age effect.ConclusionsThe present study suggests a correlation between the intrafamilial phenotypic differences observed in RTT families and their respective XCI pattern in blood, in contrast to sporadic RTT cases where a similar correlation has not been demonstrated. Furthermore, we found de novo MECP2 frameshift mutations frequently to be of paternal origin, although not with the same high paternal occurrence as in sporadic cases with C to T transitions. This suggests further investigations of more families. This study emphasizes the need for thorough genetic counselling of families with a newly diagnosed RTT patient.

Low bone turnover phenotype in Rett syndrome: results of biochemical bone marker analysis.

Background:Patients with Rett syndrome (RTT) are at risk of having low bone mass and low-energy fractures.Methods:We characterized bone metabolism by both bone formation and resorption markers in blood in a RTT population of 61 girls and women and 122 well-matched healthy controls. Levels of N-terminal propeptides of collagen type 1 (P1NP), C-terminal telopeptide cross links (CTX), osteocalcin (OC), and bone-specific alkaline phosphatase (B-ALP) were compared between RTT patients and controls in regression models adjusted for BMI, vitamin D status, volumetric bone mineral apparent density of the lumbar spine (vBMADspine), and femoral neck (vBMADneck). We examined biochemical bone marker levels overall and stratified to persons younger than age 25 y or equal to or older than age 25 y.Results:The RTT patients had reduced levels of all biochemical bone markers (P < 0.05), which remained significant in persons younger than 25 y (P ≤ 0.001) regarding P1NP, CTX, and OC. Bone marker levels were not significantly associated to methyl-CpG-binding protein 2 (MECP2) mutation group, walking ability, or previous low-energy fractures.Conclusion:Our findings of a low bone turnover state in girls with RTT suggest critical attention to medical treatment of low bone mass in young RTT patients.

Mutational and phenotypical spectrum of phenylalanine hydroxylase deficiency in Denmark

We describe the genotypes of the complete cohort, from 1967 to 2014, of phenylketonuria (PKU) patients in Denmark, in total 376 patients. A total of 752 independent alleles were investigated. Mutations were identified on 744 PKU alleles (98.9%). In total, 82 different mutations were present in the cohort. The most frequent mutation c.1315+1G>A (IVS12+1G>A) was found on 25.80% of the 744 alleles. Other very frequent mutations were c.1222C>T (p.R408W) (16.93%) and c.1241A>G (p.Y414C) (11.15%). Among the identified mutations, five mutations; c.532G>A (p.E178K), c.730C>T (p.P244S), c.925G>A (p.A309T), c.1228T>A (p.F410I), and c.1199+4A>G (IVS11+4A>G) have not been reported previously. The metabolic phenotypes of PKU are classified into four categories; ‘classical PKU’, ‘moderate PKU’, ‘mild PKU’ and ‘mild hyperphenylalaninemia’. In this study, we assigned the phenotypic outcome of three of the five novel mutations and furthermore six not previously classified mutations to one of the four PKU categories.

A thorough MECP2 mutation analysis

To the Editor: With great interest we have read the short report of Takahashi et al. entitled, ‘Skewed X chromosome inactivation failed to explain the normal phenotype of a carrier female with MECP2 mutation resulting in Rett syndrome’. The report describes a novel missense MECP2 mutation (P.A447T) detected in a girl with Rett syndrome and her asymptomatic carrier mother. Results from X-chromosome inactivation (XCI) studies performed on DNA from the mother showed a non-random XCI pattern. Further investigation showed that the mother’s predominantly active X-chromosome harbours the mutant allele. The authors speculate how these results could be correlated to the carrier mother’s normal phenotype and her daughter’s Rett phenotype. We consider that several relevant investigations are missing – especially to justify the title of this report. The authors need to address further genetic aspects before proposing a functional pathogenic nature of A447T. We lack information regarding the MECP2 mutation analysis. Was the Rett patient tested according to current knowledge about MECP2 mutations including testing for mutations in exon 1 or large deletions/duplications in the MECP2 gene? (1–4) If present, it could explain the Rett syndrome phenotype and then A447T would most likely be a rare variation. Are there any relatives in this family (siblings to the patient, to her mother or grandparents) who could be tested? Finding A447T in other family members (especially males) with normal phenotype would be a strong indication of a rare variation. What do protein substitution programs predict? Is A447T predicted to be a tolerated amino acids shift in MeCP2? It has to be taken into consideration that no other pathogenic missense mutations in the C-terminal of MeCP2 have been published in a Rett patient.