Kirstine Ravn

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.

Dimensional phenotypic analysis and functional categorisation of mutations reveal novel genotype-phenotype associations in Rett syndrome.

We aimed to improve the understanding of genotype–phenotype correlations in Rett syndrome (RS) by adopting a novel approach to categorising phenotypic dimensions – separating typicality of presentation, outcome severity and age of onset – and by classifying MECP2 mutations strictly by predicted functional attributes. MECP2 mutation screening results were available on 190 patients with a clinical diagnosis of RS (140 cases with classic RS, 50 with atypical RS). 135 cases had identified mutations. Of the 140 patients, 116 with classic RS (82.9%) had an identified mutation compared with 19 of 50 patients (38%) with an atypical presentation. Cases with early onset of regression and seizures, and those with clinical features that might indicate alternative aetiologies, were less likely to have mutations. Individuals with late truncating mutations had a less typical presentation than cases with missense and early truncating mutations, presumably reflecting greater residual function of MECP2 protein. Individuals with early truncating mutations had a more severe outcome than cases with missense and late truncating mutations. These findings held when restricting the analysis to cases over 15 years of age and classic cases only. Previous findings of variation in severity among the common mutations were confirmed. The approach to phenotypic and genotypic classification adopted here allowed us to identify genotype–phenotype associations in RS that may aid our understanding of pathogenesis and also contribute to clinical knowledge on the impact of different types of mutations.

No correlation between phenotype and genotype in boys with a truncating MECP2 mutation

Rett syndrome (RS, MIM 312750) is an X linked dominant neurodevelopmental disorder, which has been considered to affect girls only.1 Males were thought to be aborted spontaneously or to have a different phenotype.2nnThe disease is caused by mutations in MECP2 , encoding a methyl-CpG binding protein MeCP2.3 MeCP2 is an abundantly expressed protein acting as a global transcription repressor.4 The protein contains two domains, the 85 amino acid methyl-CpG binding domain (MBD) and the 102 amino acid transcriptional repression domain (TRD).4,5 Furthermore, a domain in the C terminal part of the protein, facilitating DNA binding and harbouring the nuclear localising signal (NLS), has been described.6nnThe mutation responsible for RS has now been found in the majority of RS patients and more than 100 different mutations have been published. Although some association between phenotype and genotype has been shown, their correlation is not predictive of the clinical manifestations in the individual case.7,8nnSince 1999, when mutations in MECP2 were first reported in patients with RS,3 it has been possible to analyse boys for mutations resulting in a confirmation of the diagnosis in some of the earlier reported cases.9,10nnScreening of groups of patients with mental retardation of different types has further identified MECP2 mutations in males. So far, 17 cases of males with a disease causing mutation in MECP2 have been found. However, six of them were found to have Klinefelter syndrome or were mosaics for the mutation in question.8–15 The latter can roughly be divided into two groups, the severe neonatal cases, who die within the first years of life, all caused by an MECP2 mutation that in females causes classical RS,8–10 and the non-specific mental retardation type with mutations that, if …

High Incidence of Propionic Acidemia in Greenland Is Due to a Prevalent Mutation, 1540insCCC, in the Gene for the β-Subunit of Propionyl CoA Carboxylase

Propionyl CoA carboxylase (PCC) is a mitochondrial, biotin-dependent enzyme involved in the catabolism of amino acids, odd-chain fatty acids, and other metabolites. PCC consists of two subunits, alpha and beta, encoded by the PCCA and PCCB genes, respectively. Inherited PCC deficiency due to mutations in either gene results in propionic acidemia (PA), an autosomal recessive disease. Surprisingly, PA is highly prevalent among Inuits in Greenland. We have analyzed reverse transcriptase-PCR products of the beta-subunit mRNA, to characterize the responsible mutation(s). A 3-bp insertion, 1540insCCC, was found in homozygous form in three patients and in compound heterozygous form in one patient. The resulting PCC has no measurable activity, and the mutant beta-subunit appears to be very unstable. To test the hypothesis that a common mutation is responsible for PA in the Greenlandic Inuit population, 310 anonymous DNA samples of Inuit origin were screened for 1540insCCC. We found a carrier frequency of 5%, which is very high compared with those of most other autosomal recessive diseases. Analysis of alleles of a very closely linked marker, D3S2453, revealed a high degree of linkage disequilibrium between one specific allele and 1540insCCC, suggesting that this mutation may be a founder mutation.

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

Succinate-CoA ligase deficiency due to mutations in SUCLA2 and SUCLG1: phenotype and genotype correlations in 71 patients

BackgroundThe encephalomyopathic mtDNA depletion syndrome with methylmalonic aciduria is associated with deficiency of succinate-CoA ligase, caused by mutations in SUCLA2 or SUCLG1. We report here 25 new patients with succinate-CoA ligase deficiency, and review the clinical and molecular findings in these and 46 previously reported patients.Patients and resultsOf the 71 patients, 50 had SUCLA2 mutations and 21 had SUCLG1 mutations. In the newly-reported 20 SUCLA2 patients we found 16 different mutations, of which nine were novel: two large gene deletions, a 1xa0bp duplication, two 1xa0bp deletions, a 3xa0bp insertion, a nonsense mutation and two missense mutations. In the newly-reported SUCLG1 patients, five missense mutations were identified, of which two were novel. The median onset of symptoms was two months for patients with SUCLA2 mutations and at birth for SUCLG1 patients. Median survival was 20xa0years for SUCLA2 and 20xa0months for SUCLG1. Notable clinical differences between the two groups were hepatopathy, found in 38xa0% of SUCLG1 cases but not in SUCLA2 cases, and hypertrophic cardiomyopathy which was not reported in SUCLA2 patients, but documented in 14xa0% of cases with SUCLG1 mutations. Long survival, to age 20xa0years or older, was reported in 12xa0% of SUCLA2 and in 10xa0% of SUCLG1 patients. The most frequent abnormality on neuroimaging was basal ganglia involvement, found in 69xa0% of SUCLA2 and 80xa0% of SUCLG1 patients. Analysis of respiratory chain enzyme activities in muscle generally showed a combined deficiency of complexes I and IV, but normal histological and biochemical findings in muscle did not preclude a diagnosis of succinate-CoA ligase deficiency. In five patients, the urinary excretion of methylmalonic acid was only marginally elevated, whereas elevated plasma methylmalonic acid was consistently found.ConclusionsTo our knowledge, this is the largest study of patients with SUCLA2 and SUCLG1 deficiency. The most important findings were a significantly longer survival in patients with SUCLA2 mutations compared to SUCLG1 mutations and a trend towards longer survival in patients with missense mutations compared to loss-of-function mutations. Hypertrophic cardiomyopathy and liver involvement was exclusively found in patients with SUCLG1 mutations, whereas epilepsy was much more frequent in patients with SUCLA2 mutations compared to patients with SUCLG1 mutations. The mutation analysis revealed a number of novel mutations, including a homozygous deletion of the entire SUCLA2 gene, and we found evidence of two founder mutations in the Scandinavian population, in addition to the known SUCLA2 founder mutation in the Faroe Islands.

Mutations in COA3 cause isolated complex IV deficiency associated with neuropathy, exercise intolerance, obesity, and short stature

Background We investigated a subject with an isolated cytochrome c oxidase (COX) deficiency presenting with an unusual phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature. Methods and results Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed an almost complete lack of COX assembly in subject fibroblasts, consistent with the very low enzymatic activity, and pulse-labelling mitochondrial translation experiments showed a specific decrease in synthesis of the COX1 subunit, the core catalytic subunit that nucleates assembly of the holoenzyme. Whole exome sequencing identified compound heterozygous mutations (c.199dupC, c.215A>G) in COA3, a small inner membrane COX assembly factor, resulting in a pronounced decrease in the steady-state levels of COA3 protein. Retroviral expression of a wild-type COA3 cDNA completely rescued the COX assembly and mitochondrial translation defects, confirming the pathogenicity of the mutations, and resulted in increased steady-state levels of COX1 in control cells, demonstrating a role for COA3 in the stabilisation of this subunit. COA3 exists in an early COX assembly complex that contains COX1 and other COX assembly factors including COX14 (C12orf62), another single pass transmembrane protein that also plays a role in coupling COX1 synthesis with holoenzyme assembly. Immunoblot analysis showed that COX14 was undetectable in COA3 subject fibroblasts, and that COA3 was undetectable in fibroblasts from a COX14 subject, demonstrating the interdependence of these two COX assembly factors. Conclusions The mild clinical course in this patient contrasts with nearly all other cases of severe COX assembly defects that are usually fatal early in life, and underscores the marked tissue-specific involvement in mitochondrial diseases.

Duplication of MAOA , MAOB , and NDP in a patient with mental retardation and epilepsy

Recently, Whibley et al1 reported on a family carrying a microdeletion at Xp11.3 involving only the MAOA and MAOB genes. The affected brothers in this family suffered from severe mental retardation, epilepsy, stereotypic hand movements, and lip smacking. Here we report on a male patient with mental retardation and intractable epilepsy carrying a duplication of Xp11.3 involving the MAOA, MAOB, and NDP genes.

CLPB variants associated with autosomal-recessive mitochondrial disorder with cataract, neutropenia, epilepsy, and methylglutaconic aciduria.

3-methylglutaconic aciduria (3-MGA-uria) is a nonspecific finding associated with mitochondrial dysfunction, including defects of oxidative phosphorylation. 3-MGA-uria is classified into five groups, of which one, type IV, is genetically heterogeneous. Here we report five children with a form of type IV 3-MGA-uria characterized by cataracts, severe psychomotor regression during febrile episodes, epilepsy, neutropenia with frequent infections, and death in early childhood. Four of the individuals were of Greenlandic descent, and one was North American, of Northern European and Asian descent. Through a combination of homozygosity mapping in the Greenlandic individuals and exome sequencing in the North American, we identified biallelic variants in the caseinolytic peptidase B homolog (CLPB). The causative variants included one missense variant, c.803C>T (p.Thr268Met), and two nonsense variants, c.961A>T (p.Lys321*) and c.1249C>T (p.Arg417*). The level of CLPB protein was markedly decreased in fibroblasts and liver of affected individuals. CLPB is proposed to function as a mitochondrial chaperone involved in disaggregation of misfolded proteins, resulting from stress such as heat denaturation.

X-linked congenital ptosis and associated intellectual disability, short stature, microcephaly, cleft palate, digital and genital abnormalities define novel Xq25q26 duplication syndrome

Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76xa0Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1,RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.