Karen Helene Ørstavik

Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EMD) is a condition characterized by the clinical triad of early-onset contractures, progressive weakness in humeroperoneal muscles, and cardiomyopathy with conduction block. The disease was described for the first time as an X-linked muscular dystrophy, but autosomal dominant and autosomal recessive forms were reported. The genes for X-linked EMD and autosomal dominant EMD (AD-EMD) were identified. We report here that heterozygote mutations in LMNA, the gene for AD-EMD, may cause diverse phenotypes ranging from typical EMD to no phenotypic effect. Our results show that LMNA mutations are also responsible for the recessive form of the disease. Our results give further support to the notion that different genetic forms of EMD have a common pathophysiological background. The distribution of the mutations in AD-EMD patients (in the tail and in the 2A rod domain) suggests that unique interactions between lamin A/C and other nuclear components exist that have an important role in cardiac and skeletal muscle function.

Another Case of Imprinting Defect in a Girl with Angelman Syndrome Who Was Conceived by Intracytoplasmic Sperm Injection

To the Editor: Intracytoplasmic sperm injection (ICSI) has been established as an efficient treatment for male infertility and also as a supplement to in vitro fertilization (IVF) without obvious male infertility. ICSI is now regarded as a procedure that is safe overall, and no increase in developmental delay was found in a follow-up of 221 ICSI-conceived children in the 2nd year of life (Sutcliffe et al. 2001). However, the possibility of an increased risk of imprinting defects has been raised (Manning et al. 2000). Two children conceived by ICSI who had Angelman syndrome (AS [MIM 105830]) due to a presumably sporadic imprinting defect have recently been reported (Cox et al. 2002). We here report a 3.5-year-old girl with AS due to a sporadic imprinting defect, born of a pregnancy that was also the result of ICSI. The girl was the first child of a 35-year-old mother and a 36-year-old father. The father has a healthy daughter by another partner, and sperm analysis was normal on three different occasions. The mother had one spontaneous abortion and two extrauterine pregnancies before treatment with IVF. Traditional IVF did not result in fertilized eggs, and ICSI was therefore performed in spite of the normal sperm analysis of the father. The first ICSI pregnancy resulted in another spontaneous abortion, whereas the second ICSI procedure resulted in a normal pregnancy. Birth was at term, birth weight was 3,760 g, length was 54 cm, and head circumference was 36 cm (75th percentile). Development was considered normal for the first 3–4 mo, after which she started to have infections. She walked at age 2 years. She had no epilepsy but had an abnormal electroencephalogram with large-amplitude slow-spike waves. There was no language development. Chromosomes, including subtelomeres, were normal. At age 3 years, her height and weight were at the 50th percentile, whereas her head circumference was 1 cm below the 2.5th percentile. She was mentally retarded and atactic. She was dysmorphic, with a square face, deep-set eyes, and a protruding tongue. FISH analysis using the SNRPN probe (MIM 182279), as well as microsatellite studies, revealed normal chromosomes 15 of biparental origin. A common large deletion of 15q11-q13 and uniparental paternal disomy could therefore be excluded. Methylation-specific Southern blot analysis and methylation-specific PCR (Zeschnigk et al. 1997) for the SNRPN locus showed the presence of a normal unmethylated paternal band and the complete absence of a methylated maternal band, indicating that the patient had an imprinting defect. Quantitative Southern blot analysis of the critical AS imprinting center (IC) region (Buiting et al. 1999) showed a normal dosage; therefore, an IC deletion was unlikely. This result was confirmed by sequence analysis of the 880-bp AS-IC element, where the patient was heterozygous for three different SNPs. Both parents had normal chromosomes and a normal methylation pattern. These findings suggest that the patient belongs to the group of patients with a sporadic imprinting defect (Buiting et al. 1998). Both patients reported by Cox et al. (2002) had fathers with sperm abnormalities, and the possibility that the imprinting defect could be related to male infertility was discussed. The father of the child reported here had normal sperm, and a relationship to male infertility is therefore unlikely. However, similar to the mother, the maternal grandmother also had a history of reproductive difficulties. In addition to three healthy children, she had four spontaneous abortions and one daughter who was stillborn at term. Since both the maternal grandmother and the mother had reproductive difficulties, a maternal oogenesis defect cannot be excluded. A sporadic imprinting defect is a very rare cause of AS, and Cox et al. (2002) therefore considered a relationship to the ICSI procedure to be likely. The report of a third patient with this rare disorder further supports the assumption that ICSI can lead to an increased risk for imprinting defects.

Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans

Remodeling of the cytoskeleton is central to the modulation of cell shape and migration. Filamin A, encoded by the gene FLNA, is a widely expressed protein that regulates re-organization of the actin cytoskeleton by interacting with integrins, transmembrane receptor complexes and second messengers. We identified localized mutations in FLNA that conserve the reading frame and lead to a broad range of congenital malformations, affecting craniofacial structures, skeleton, brain, viscera and urogenital tract, in four X-linked human disorders: otopalatodigital syndrome types 1 (OPD1; OMIM 311300) and 2 (OPD2; OMIM 304120), frontometaphyseal dysplasia (FMD; OMIM 305620) and Melnick–Needles syndrome (MNS; OMIM 309350). Several mutations are recurrent, and all are clustered into four regions of the gene: the actin-binding domain and rod domain repeats 3, 10 and 14/15. Our findings contrast with previous observations that loss of function of FLNA is embryonic lethal in males but manifests in females as a localized neuronal migration disorder, called periventricular nodular heterotopia (PVNH; refs. 3–6). The patterns of mutation, X-chromosome inactivation and phenotypic manifestations in the newly described mutations indicate that they have gain-of-function effects, implicating filamin A in signaling pathways that mediate organogenesis in multiple systems during embryonic development.

A homeobox gene, HLXB9, is the major locus for dominantly inherited sacral agenesis

Partial absence of the sacrum is a rare congenital defect which also occurs as an autosomal dominant trait; association with anterior meningocoele, presacral teratoma and anorectal abnormalities constitutes the Currarino triad (MIM 176450). Malformation at the caudal end of the developing notochord at approximately Carnegie stage 7 (16 post-ovulatory days), which results in aberrant secondary neurulation, can explain the observed pattern of anomalies. We previously reported linkage to 7q36 markers in two dominantly inherited sacral agenesis families. We now present data refining the initial subchromosomal localization in several additional hereditary sacral agenesis (HSA) families. We excluded several candidate genes before identifying patient-specific mutations in a homeobox gene, HLXB9, which was previously reported to map to 1q41-q42.1 and to be expressed in lymphoid and pancreatic tissues.

RAB23 Mutations in Carpenter Syndrome Imply an Unexpected Role for Hedgehog Signaling in Cranial-Suture Development and Obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.

Genotypic and Phenotypic Spectrum in Tricho-Rhino-Phalangeal Syndrome Types I and III

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities. Three subtypes have been described: TRPS I, caused by mutations in the TRPS1 gene on chromosome 8; TRPS II, a microdeletion syndrome affecting the TRPS1 and EXT1 genes; and TRPS III, a form with severe brachydactyly, due to short metacarpals, and severe short stature, but without exostoses. To investigate whether TRPS III is caused by TRPS1 mutations and to establish a genotype-phenotype correlation in TRPS, we performed extensive mutation analysis and evaluated the height and degree of brachydactyly in patients with TRPS I or TRPS III. We found 35 different mutations in 44 of 51 unrelated patients. The detection rate (86%) indicates that TRPS1 is the major locus for TRPS I and TRPS III. We did not find any mutation in the parents of sporadic patients or in apparently healthy relatives of familial patients, indicating complete penetrance of TRPS1 mutations. Evaluation of skeletal abnormalities of patients with TRPS1 mutations revealed a wide clinical spectrum. The phenotype was variable in unrelated, age- and sex-matched patients with identical mutations, as well as in families. Four of the five missense mutations alter the GATA DNA-binding zinc finger, and six of the seven unrelated patients with these mutations may be classified as having TRPS III. Our data indicate that TRPS III is at the severe end of the TRPS spectrum and that it is most often caused by a specific class of mutations in the TRPS1 gene.

Twin study of genetic and aging effects on X chromosome inactivation

To investigate the genetic influence on X chromosome inactivation and on age-related skewing of X inactivation, in particular, we analysed the X inactivation pattern (XIP) in peripheral blood cells from 118 young monozygotic (MZ) twin pairs (18–53 years), 82 elderly MZ twin pairs (55–94 years), 146 young dizygotic (DZ) twin pairs (20–54 years) and 112 elderly DZ twin pairs (64–95 years). Elderly twins had a higher frequency of skewed X inactivation (34%) than young twins (15%) (P<0.001). Our data suggest that the increase in skewing occurs after age 50–60 years. The intraclass correlation was 0.61 and 0.58 in young and elderly MZ twin pairs, and 0.08 and 0.09 in young and elderly DZ twin pairs. Biometric analysis showed that dominant genetic effects accounted for 63 and 58% of the variance of XIP in the young and elderly twin pairs, respectively. The dominant genetic effect and the shared environment for monochorionic MZ twins may explain the high intraclass correlation for the MZ twin pairs compared to the DZ twin pairs. We did not observe a significant decrease in the intraclass correlation in elderly MZ twins compared to young MZ twins, which would be expected if age-related skewing were due to stochastic factors. We conclude that the increased skewing with age implies that a genetically dependent selection of blood cells take place.

High frequency of skewed X inactivation in young breast cancer patients

Introduction: Patients with invasive ovarian cancer were recently shown to have a higher frequency of skewed X chromosome inactivation in peripheral blood cells compared to patients with borderline cancer and controls. In this study, we analysed the X inactivation pattern in peripheral blood from 216 breast cancer patients. Methods: X inactivation analysis was performed using HpaII predigestion of DNA followed by PCR of the highly polymorphic CAG repeat of the androgen receptor gene (AR), which amplifies the undigested inactive X chromosome only. The X inactivation pattern was classified as skewed when 90% or more of the cells preferentially used one X chromosome. Results: Young breast cancer patients (27-45 years) had a higher frequency of skewed X inactivation than young controls (13 and 1%, respectively) (p=0.009), whereas no difference was found for middle aged and older patients compared to controls of a similar age. Conclusions: A germline mutation in an X linked tumour suppressor gene may give a proliferative advantage to cells with this mutation on the active X chromosome, thus causing skewed X inactivation and an increased risk for developing cancer. Another possible explanation could be that females with a constitutionally skewed X inactivation pattern are more susceptible to develop breast cancer because of an X linked low penetrance susceptibility allele that is affected by the inactivation pattern.

X chromosome inactivation in clinical practice

X chromosome inactivation (XCI) is the transcriptional silencing of the majority of genes on one of the two X chromosomes in mammalian females. Females are, therefore, mosaics for two cell lines, one with the maternal X and one with the paternal X as the active chromosome. The relative proportion of the two cell lines, the X inactivation pattern, may be analyzed by simple assays in DNA from available tissues. This review focuses on medical issues related to XCI in X-linked disorders, and on the value of X inactivation analysis in clinical practice.X chromosome inactivation (XCI) is the transcriptional silencing of the majority of genes on one of the two X chromosomes in mammalian females. Females are, therefore, mosaics for two cell lines, one with the maternal X and one with the paternal X as the active chromosome. The relative proportion of the two cell lines, the X inactivation pattern, may be analyzed by simple assays in DNA from available tissues. This review focuses on medical issues related to XCI in X-linked disorders, and on the value of X inactivation analysis in clinical practice.

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.