A new highly penetrant form of obesity due to deletions on chromosome 16p11.2.

Obesity has become a major worldwide challenge to public health, owing to an interaction between the Western 'obesogenic' environment and a strong genetic contribution. Recent extensive genome-wide association studies (GWASs) have identified numerous single nucleotide polymorphisms associated with obesity, but these loci together account for only a small fraction of the known heritable component. Thus, the 'common disease, common variant' hypothesis is increasingly coming under challenge. Here we report a highly penetrant form of obesity, initially observed in 31 subjects who were heterozygous for deletions of at least 593 kilobases at 16p11.2 and whose ascertainment included cognitive deficits. Nineteen similar deletions were identified from GWAS data in 16,053 individuals from eight European cohorts. These deletions were absent from healthy non-obese controls and accounted for 0.7% of our morbid obesity cases (body mass index (BMI) >or= 40 kg m(-2) or BMI standard deviation score >or= 4; P = 6.4 x 10(-8), odds ratio 43.0), demonstrating the potential importance in common disease of rare variants with strong effects. This highlights a promising strategy for identifying missing heritability in obesity and other complex traits: cohorts with extreme phenotypes are likely to be enriched for rare variants, thereby improving power for their discovery. Subsequent analysis of the loci so identified may well reveal additional rare variants that further contribute to the missing heritability, as recently reported for SIM1 (ref. 3). The most productive approach may therefore be to combine the 'power of the extreme' in small, well-phenotyped cohorts, with targeted follow-up in case-control and population cohorts.

Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients.

MECP2 mutations are identifiable in approximately 80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.

Hearing improvement in patients with Fabry disease treated with agalsidase alfa.

AIM: To describe the nature and prevalence of hearing loss in Fabry disease, and its response to enzyme replacement therapy (ERT) with agalsidase alfa. METHODS: Fifteen male patients with Fabry disease were enrolled in a randomized, double-blind study and received placebo (n = 8) or ERT (n = 7) with agalsidase alfa for 6 months. This was followed by an open-label extension of 36 months thus far. Alongside this trial, an additional eight men and two women have so far received open-label ERT for between 6 and 30 months. Pure-tone audiometry, impedance audiometry and otoacoustic emission testing were performed at 0 (baseline), 6, 18, 30 and 42 months. RESULTS: Nine patients (36%) had bilateral and ten (40%) had unilateral high-frequency sensorineural hearing loss (SNHL). Three (12%) had unilateral middle ear effusions with conductive losses persisting beyond 6 months. Only five patients (20%) had normal hearing. The high-frequency SNHL deteriorated over the first 6 months in both placebo and active treatment groups by a median 6.3 dB (p < 0.0001, Wilcoxon matched-pairs). This hearing loss subsequently improved above baseline by 1.5 dB at 18 months (p = 0.07), by 5.0 dB at 30 months (p = 0.006) and by 4.0 dB at 42 months (p = 0.01). CONCLUSION: Significant hearing loss, usually high-frequency SNHL, is a common manifestation of Fabry disease in adults. Alpha-galactosidase A replacement therapy with agalsidase alfa appears to reverse the hearing deterioration in these patients. This improvement, however, is gradual, suggesting the need for long-term ERT.

Assessment of health-related quality-of-life in males with Anderson Fabry Disease before therapeutic intervention.

Anderson Fabry Disease (AFD) is an extremely painful and debilitating multi-system X-linked disorder due to alpha-galactosidase enzyme deficiency. To date, no baseline data on health-related quality-of-life (HR-QoL) have been reported in males affected with this condition. In this study, 38 males with AFD completed Medical Outcomes Study Short Form, EuroQoL questionnaires and an AFD-specific questionnaire prior to the start of a trial involving replacement therapy with alpha-galactosidase. Results from these questionnaires were compared to the results from a similar HR-QoL study in males with severe haemophilia (factor VIII/IX deficiency) that used the same questionnaires and to the results of two large normative studies. The results on both questionnaires showed that in most instances males with AFD recorded significantly lower HR-QoL compared with males in the general population and individuals with severe haemophilia after adjusting for differences in age. These findings suggest therefore, that the scope for improvement in HR-QoL as a result of treatment with an appropriate agent is extremely large.

Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males.

OBJECTIVES: To determine the natural history of Anderson-Fabry disease (AFD) as a baseline for efficacy assessment of potentially therapeutic drugs. DESIGN: The first large cross sectional study of a patient cohort from the AFD clinical and genetic register (UK), maintained for the last 15 years. MEASURES: Prevalence, mortality, frequency of AFD manifestations, and impact of disease on patient lives, assessed from the AFD register and the disease specific questionnaire. RESULTS: The median cumulative survival was 50 years (n=51), which represents an approximately 20 year reduction of life span. Neuropathic pain was present in 77% (n=93) with mean pain score of 5 (scale 0-10) despite treatment with anticonvulsants and opiates. Pain stopped in only 11%. Cerebrovascular complications developed in 24.2% and renal failure in 30%. The onset and progression of serious AFD manifestations was highly variable. The relationship of gastrointestinal manifestations on weight, using body mass index (BMI), was significant (p=0.01). High frequency sensorineural deafness was confirmed in 78% of audiograms. Neuropathic pain and angiokeratoma were absent in five adult males (approximately 5%). Median age at diagnosis of AFD was 21.9 years (n=64). IMPACT OF DISEASE: Attendance at school, sports, and social activity were significantly affected by AFD. Only 56.6% (n=46) of patients were employed. Psychosexual effects of genital angiokeratoma, genital pain, and impotence were not previously recognised. CONCLUSION: The majority of males experience multiple disease manifestations and the duration of neuropathic pain was lifelong. The AFD register proved useful for the determination of baseline disease parameters in this cohort.

Neuropathic pain in Anderson-Fabry disease: pathology and therapeutic options.

An inherited deficiency of the enzyme alpha-galactosidase A is manifest clinically as Anderson-Fabry disease. Most affected patients present with severe peripheral pain in childhood or early adult life, and frequently progress to multi-organ failure by the 4th or 5th decades. The present review examines the probable mechanisms that contribute to pain in these patients, and outlines some of the treatment options that are currently used. The successful outcome of the first two trials of enzyme replacement therapy suggest that this disease might be amenable in the future to gene therapy.

Apparent normalisation of fetal renal size in autosomal dominant polycystic kidney disease (PKD1).

We present a family with adult onset autosomal dominant polycystic kidney disease (ADPKD) in two generations, linked to the PKD1 locus and with paternal transmission to the fetus. The fetus carried the PKD1 haplotype and was, therefore a gene carrier. Progressive hyperechogenic renal enlargement, but no cysts, was documented by serial fetal ultrasounds at 21, 23 and 34 weeks of gestation. Surprisingly, the newborn renal scan showed normal sized kidneys with apparently normal corticomedullary differentiation. However, at 11 months of age, the evolution of cysts in one kidney, and then in the other kidney at 20 months, was documented by ultrasound in the absence of clinical symptoms or signs. The observed normalisation of fetal renal ultrasound appearances at birth has not previously been described in fetuses presenting with PKD1.

Prenatal diagnosis of autosomal dominant polycystic kidney disease (PKD1) presenting in utero and prognosis for very early onset disease.

We describe four prenatal diagnoses in a family with autosomal dominant polycystic kidney disease. Two pregnancies were terminated following the detection of enlarged echogenic fetal kidneys with cysts. Histopathological examination confirmed the diagnosis of polycystic kidney disease. Linkage to PKD1 was obtained by the analysis of DNA from relatives in three generations and from paraffin blocks and formalin fixed fetal tissues. Prenatal DNA analysis in subsequent pregnancies identified one unaffected fetus and one fetus carrying the high risk PKD1 allelle. Information on survival and subsequent outcome of PKD cases presenting in utero was requested by this family before prenatal testing was performed. Of 83 reported cases of ADPKD presenting in utero (excluding termination of pregnancy) or in the first few months of life, 43% died before 1 year. Longitudinal follow up of 24 children in two studies showed that 67% of survivors developed hypertension, of whom three had end stage renal failure at a mean age of 3 years.

A novel acid alpha-glucosidase mutation identified in a Pakistani family with glycogen storage disease type II.

A novel mutation, C118T, in exon 2 of the acid alpha-glucosidase gene has been found in an infant with glycogen storage disease type II. This mutation is predicted to result in protein truncation. The phenotype was that of the severe infantile form of the disorder with lack of motor development, but with eye regard, social smile and vocalization. The parents were heterozygous for C118T and belong to an Islamic community opposed to termination of pregnancy. As the C118T mutation results in the loss of one of two AvaI sites present in an informative PCR product, reliable premarriage carrier detection became possible and was acceptable to the members of this extended family.

Gene transfer and expression of human alpha-galactosidase from mouse muscle in vitro and in vivo.

Lysosomal storage disorders are amenable to treatment by enzyme replacement. Genetic modification of muscle via direct injection of expression vectors might represent an alternative method of providing the defective enzymes, if adequate and long-lasting expression levels can be achieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme alpha-galactosidase (alpha-gal). In differentiated myotubes, a construct containing the myosin light chain 1/3 enhancer in combination with the human cytomegalovirus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit beta-myosin heavy chain promoter, or containing the CMV promoter only. Increased enzymatic activity was detectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in alpha-gal were able to take up the enzyme from medium conditioned by transfected myoblasts. This did not occur in the presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the first report in which a correctly processed form of human alpha-gal was expressed and secreted from differentiated muscle cells. Direct injection of a plasmid expression vector into mouse tibialis anterior muscle showed significantly increased levels of alpha-gal 7 days after injection.

Gene therapy: panacea or placebo? I. Strategies and limitations of gene therapy.

Progress in genetics and molecular medicine has led to characterization of many disorders at the level of specific genes. Techniques for reliable diagnosis of these disorders have been developed in parallel. Attempts are currently being made to develop DNA-based therapeutic procedures to correct genetic diseases. These procedures are known under a common name of gene therapy. The initial step in gene therapy is the delivery of the gene of interest into target cells. There are several conceptually different approaches to achieve this, e.g. targeting can be accomplished by using viral vectors (transduction) or DNA-mediated routes (transformation) either ex vivo or in vivo. The selection of vector systems and the choice of delivering genes either into isolated cells or directly in the organism depend on factors like: the nature of target cell or organ, the levels of expression required, stability and/or regulation of expression, safety, etc. Many viruses have been adapted for use as vectors for gene therapy, according to their specific properties. Viral genomes have been modified to remove their ability to replicate and to increase the cloning capacity. Non-viral gene delivery systems rely on cellular mechanisms to import DNA into the cell nucleus and different methods to enhance DNA uptake have been attempted. Despire many significant achievements there are still obstacles to the development of effective clinical products. Most significant are the low levels and stability of expression of introduced genes and immune responses to vectors and/or gene products.

Gene therapy: panacea or placebo? II. Main applications of gene therapy.

For most disorders the ideal goal of gene therapy is the repair of the mutated gene in the target tissue. However, the techniques required for such an approach are still at an early stage of development. Most current research is directed towards delivery of normal gene sequences in order to generate active protein and compensate for the lack of endogenous production. This approach may be suitable for the treatment of recessive monogenic disorders, but is inappropriate for dominantly inherited disorders. Therefore, gene therapy was originally intended as a most promising approach for the treatment of inborn errors of metabolism. However, apart from the correction of heritable genetic disorders, gene delivery has many potential applications including treatment of malignancies, atherosclerosis and vascular proliferative disorders, rheumatoid arthritis and viral infections. The authors discuss significant examples marking progress in the development of gene therapy for specific diseases, present some hopes and hurdles that have arisen from some recent preclinical and early clinical trials.

Punctate palmoplantar keratoderma and malignancy in a four-generation family.

We report a large kindred in which a punctate palmoplantar keratoderma (PPK) is associated with malignancy, including Hodgkin's disease, renal, breast, pancreatic and colonic adenocarcinomas. The family was traced through four generations, and over 320 individuals were identified, of whom 49 had punctate PPK. The punctate PPK appeared to be inherited as an autosomal dominant trait with variable penetrance. Ten of the 43 adults (23%) with punctate PPK developed malignancies, and five of these developed before the age of 50. Of the 271 unaffected individuals, six (2%) have developed malignancies, one prior to the age of 50. The association of keratoderma and malignancy is discussed.

Osteopenia, abnormal dentition, hydrops fetalis and communicating hydrocephalus.

We describe a single male infant who developed severe hydrops fetalis between 19 and 28 weeks of gestation. After delivery at 32 weeks he was treated by hemofiltration, prolonged ventilation and intravenous feeding. He had hypertelorism, orbital hypoplasia without proptosis, brachydactyly, frontal and temporal bossing of the skull, central hypotonia, communicating hydrocephalus, and severe delay in psychomotor development. Signs of connective tissue disorder included: osteopenia, pathological fracture, yellow/grey discolored teeth, blue sclerae and easy bruising. Laboratory investigations failed to reveal the cause of fetal hydrops or collagen abnormality. His mother and one sib had learning difficulties. Although some of these findings may be due to perinatal factors, the connective tissue abnormalities suggest a genetic syndrome in the heterogeneous group of osteogenesis imperfecta. This case either represents the more severe end of the spectrum of Type IV osteogenesis imperfecta or the mild end of the spectrum of Cole-Carpenter syndrome.

Editing of human alpha-galactosidase RNA resulting in a pyrimidine to purine conversion.

During a study of the gene coding for alpha-galactosidase (EC 3.2.1.22), the lysosomal enzyme deficient in Fabry's disease, RT-PCR amplification of alpha-galactosidase mRNAs obtained from three different tissues isolated from males revealed a substantial number of clones with a U to A conversion at the nucleotide position 1187. Such a modification of the coding sequence would result in an amino acid substitution in the C-terminal region (Phe396Tyr) of the enzyme. Neither PCR analysis of the genomic sequence nor the RT-PCR amplification of RNA obtained by in vitro transcription of the wild-type cDNA showed this change in the sequence. Multiple genes, pseudogenes are allelic variants were excluded. Hence, we propose RNA editing as a mechanism responsible for this base change in the alpha-galactosidase RNA.

Short stature/short limb skeletal dysplasia with severe combined immunodeficiency and bowing of the femora: report of two patients and review.

We report two patients with severe combined immunodeficiency and short stature/short limb skeletal dysplasia. Case 1 presented at birth with rhizomelic shortening of the extremities and bowing of the femora. She developed clinical signs of severe combined immunodeficiency at 13 months and died at 21 months. Case 2 had severe prenatal shortening and bowing of the extremities and a small, malformed chest. Symptoms of severe combined immunodeficiency and severe failure to thrive developed soon after birth and she died at 5 months. The diagnosis of severe combined immunodeficiency in our patients was based on their clinical course and necropsy findings, supported in case 1 by the results of immune function tests. The results of investigation of immune function (immunoglobulins, lymphocyte subpopulations, lymphocyte function) are very variable in this syndrome as in other variants of severe combined immunodeficiency. Bone histopathology in both patients showed grossly irregular costochondral junctions, but normal transition of proliferating to hypertrophic chondrocytes. These cases belong to early lethal type 1 short limb skeletal dysplasia with severe combined immunodeficiency. Review of previously published cases with severe combined immunodeficiency and well documented skeletal findings show eight patients with prenatal onset of bowing and shortening of the extremities and metaphyseal abnormalities. These include two sib pairs concordant for the skeletal changes. In these cases, adenosine deaminase levels were not reported. An additional four published cases with associated adenosine deaminase deficiency had only mild metaphyseal abnormalities, but subsequently showed no linear growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Oculofacialbulbar palsy in mother and son: review of 26 reports of familial transmission within the 'Möbius spectrum of defects'.

We report a mother and son with 5th, 6th, 7th, and bulbar cranial nerve paralysis, who had two similarly affected relatives. None of them had primary skeletal defects. Twenty-six previous reports of familial cases within the heterogeneous 'Möbius spectrum of defects' were reviewed. When cranial nerve palsies were associated with a primary skeletal defect, familial transmission was not found. No recurrence was noted in 31 cases with cranial nerve palsies associated with oral abnormalities and limb defects. The term Möbius syndrome should be restricted to cases with congenital 6th and 7th nerve paralysis with skeletal defects, who have a low recurrence risk (2%). The features in an index case which may indicate a higher risk of recurrence are the absence of skeletal defects, isolated facial palsy, deafness, ophthalmoplegia, and digital contractures. A recurrence risk of 25 to 30% in these cases appears reasonable.