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Williams-Beuren syndrome (WBS, MIM 194050) is a rare condition, with striking physical and behavioural features,1–3 which occurs in 1/20 000-1/50 000 live births. Cases are generally sporadic; however, familial cases with an autosomal dominant mode of inheritance have been reported. It results in a complex phenotype with physical, cognitive, and behavioural aspects that include an uneven cognitive profile (WBSCP), with verbal tasks outstripping spatial tasks, and overall IQs in the 50-60 range. Physically, WBS phenotypes include a dysmorphic face, congenital heart disease (typically supravalvular aortic stenosis (SVAS)), growth retardation, hyperacusis, premature ageing, and often infantile hypercalcaemia. These features are caused by deletion of the Williams-Beuren syndrome critical region (WBSCR) at chromosomal position 7q11.23 on either the maternal or paternal chromosome 7. The deletion is thought to arise from recombination between misaligned repeat sequences flanking the WBSCR during meiosis. The breakpoints cluster within these repeat regions, so that most WBS patients have similar deletions of approximately 1.5 Mb. A few WBS patients have, however, been reported with smaller deletions (

Marfan syndrome (MFS) is one of the major heritable disorders of connective tissue with a prevalence of between 1 in 5-10 000.1,2 It is characterised by features in the cardiovascular, ocular, and musculoskeletal systems and the Ghent criteria form a useful framework for its diagnosis.3 Mutations in FBN1 encoding the extracellular matrix protein fibrillin-1 classically cause MFS.4 Fibrillin-1, comprising multiple repeating subunits, of which the most common is the calcium binding epidermal growth factor (cbEGF) domain, is a key component of 10-12 nm microfibrils.5 Mutation detection has not been performed routinely in MFS because of the size and complexity of FBN1, which contains 65 exons extending over 200 kb of genomic DNA.5,6 Mutations are nearly always specific to each family and to date ∼300 mutations have been reported.7–10 FBN1 mutations have been found in 20-80% of patients with MFS depending upon the clinical selection of patients and the mutation detection method used.11–17 FBN1 mutations have been reported in a wide range of phenotypes in the Marfan spectrum1; neonatal MFS is associated with some FBN1 mutations in exons 24-3218; mutations of various types throughout FBN1 have been associated with classical MFS8; and FBN1 mutations have also been reported in many of the non-classical Marfan related phenotypes.1 The Ghent criteria propose that an FBN1 mutation is only of help diagnostically if it has been previously found in a person who independently meets the criteria for diagnosis of MFS.3 Other than mutations causing neonatal MFS, which occur in exons 24-32, correlation between genotype and phenotype is poor. The majority of patients with cysteine substitutions have classical MFS, and cysteine substitutions in exons 26-32 appear to be associated with classical disease manifesting early in life. …

BACKGROUND: Inositol polyphosphate phosphatase-like 1 (INPPL1, SHIP2) is a negative regulator of insulin signalling and has previously been found to be associated with hypertension, obesity and type 2 diabetes in a cohort of families with diabetes in the UK presenting features of metabolic syndrome. In particular, a haplotype of three genetic polymorphisms (rs2276047, rs9886 and an insertion/deletion polymorphism in intron 1) was found to be strongly associated with increased susceptibility to hypertension. OBJECTIVE AND METHODS: To assess if INPPL1 variants play a direct role in the development of essential hypertension, we genotyped the three previously associated INPPL1 polymorphisms in a cohort of 712 families with severe hypertension from the BRIGHT study transmission disequilibrium test cohort. RESULTS: We found no evidence of significant association between hypertension and any of the three INPPL1 polymorphisms or haplotypes (p>0.1). CONCLUSION: These results suggest that INPPL1 variants may be involved in mechanisms causing hypertension in metabolic syndrome patients specifically.

Aims: To determine the relative frequency of mutations in three different genes (low-density lipoprotein receptor (LDLR), APOB, PCSK9) , and to examine their effect in development of coronary heart disease (CHD) in patients with clinically defined definite familial hypercholesterolaemia in UK. Patients and methods: 409 patients with familial hypercholesterolaemia patients (158 with CHD) were studied. The LDLR was partially screened by single-strand conformational polymorphism (SSCP) (exons 3, 4, 6–10 and 14) and by using a commercial kit for gross deletions or rearrangements. APOB (p.R3500Q) and PCSK9 (p.D374Y) were detected by specific assays. Coding exons of PCSK9 were screened by SSCP. Results: Mutations were detected in 253 (61.9%) patients: 236 (57.7%) carried LDLR , 10 (2.4%) carried APOB p.Q3500 and 7 (1.7%) PCSK9 p.Y374. No additional mutations were identified in PCSK9 . After adjusting for age, sex, smoking and systolic blood pressure, compared to those with no detectable mutation, the odds ratio of having CHD in those with an LDLR mutation was 1.84 (95% CI 1.10 to 3.06), for APOB 3.40 (0.71 to 16.36), and for PCSK9 19.96 (1.88 to 211.5; p = 0.001 overall). The high risk in patients carrying LDLR and PCSK9 p.Y374 was partly explained by their higher pretreatment cholesterol levels ( LDLR , PCSK9 and no mutation, 10.29 (1.85), 13.12 and 9.85 (1.90) mmol/l, respectively, p = 0.001). The post-statin treatment lipid profile in PCSK9 p.Y374 carriers was worse than in patients with no identified mutation (LDL-C, 6.77 (1.82) mmol/l v 4.19 (1.26) mmol/l, p = 0.001, HDL-C 1.09 (0.27) mmol/l v 1.36 (0.36) mmol/l, p = 0.03). Conclusions: The higher CHD risk in patients carrying PCSK9 p.Y347 or a detected LDLR mutation supports the usefulness of DNA testing in the diagnosis and management of patients with familial hypercholesterolaemia. Mutations in PCSK9 appear uncommon in patients with familial hypercholesterolaemia in UK.

BACKGROUND: A polymorphism in exon 4 (C77G) of CD45 that alters CD45 splicing has been associated with autoimmune and infectious diseases in humans. OBJECTIVE: To investigate the effect of C77G in hepatitis C virus (HCV) infected individuals and study the phenotype and function of peripheral blood mononuclear cells (PBMC) from healthy and hepatitis C infected C77G carriers. RESULTS: C77G individuals showed an increased proportion of primed CD45RA and effector memory CD8 T cells and more rapid activation of the lymphocyte specific protein tyrosine kinase (Lck) following CD3 stimulation. Transgenic mice with CD45 expression mimicking that in human C77G variants had more activated/memory T cells, more rapid proliferative responses, and activation of Lck. CONCLUSIONS: Changes in CD45 isoform expression can alter immune function in human C77G variants and CD45 transgenic mice. The C77G allele may influence the outcome of HCV infection.

Background: Spinal muscular atrophy (SMA) is the most common inherited lethal disease of children. Various genetic deletions involving the bi-allelic loss of SMN1 exon 7 are reported to account for 94% of affected individuals. Published literature places the carrier frequency for SMN1 mutations between 1 in 25 and 1 in 50 in the general population. Although SMA is considered to be a pan-ethnic disease, carrier frequencies for many ethnicities, including most ethnic groups in North America, are unknown. Objectives and methods: To provide an accurate assessment of SMN1 mutation carrier frequencies in African American, Ashkenazi Jewish, Asian, Caucasian, and Hispanic populations, more than 1000 specimens in each ethnic group were tested using a clinically validated, quantitative real-time polymerase chain reaction (PCR) assay that measures exon 7 copy number. Results: The observed one-copy genotype frequency was 1 in 37 (2.7%) in Caucasian, 1 in 46 (2.2%) in Ashkenazi Jew, 1 in 56 (1.8%) in Asian, 1 in 91 (1.1%) in African American, and 1 in 125 (0.8%) in Hispanic specimens. Additionally, an unusually high frequency of alleles with multiple copies of SMN1 was identified in the African American group (27% compared to 3.3–8.1%). This latter finding has clinical implications for providing accurate adjusted genetic risk assessments to the African American population. Conclusions: Differences in the frequency of SMA carriers were significant among several ethnic groups. This study provides an accurate assessment of allele frequencies and estimates of adjusted genetic risk that were previously unavailable to clinicians and patients considering testing.

Background: Recently the genotype/phenotype map of Wolf-Hirschhorn syndrome (WHS) has been refined, using small 4p deletions covering or flanking the critical region in patients showing only some of the WHS malformations. Accordingly, prenatal-onset growth retardation and failure to thrive have been found to result from haploinsufficiency for a 4p gene located between 0.4 and 1.3 Mb, whereas microcephaly results from haploinsufficiency of at least two different 4p regions, one of 2.2–2.38 Mb and a second one of 1.9–1.28 Mb. Methods and Results: We defined the deletion size of a ring chromosome (r(4)) in a girl with prenatal onset growth retardation, severe failure to thrive and true microcephaly but without the WHS facial gestalt and mental retardation. A high-resolution comparative genome hybridisation array revealed a 760 kb 4p terminal deletion. Conclusions: This case, together with a familial 4p deletion involving the distal 400 kb reported in normal women, may narrow the critical region for short stature on 4p to 360–760 kb. This region is also likely to contain a gene for microcephaly. “In silico” analysis of all genes within the critical region failed to reveal any strikingly suggestive expression pattern; all genes remain candidates for short stature and microcephaly.

Introduction: Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with maternal inheritance. Due to a high transmission risk and the absence of therapy in these disorders, “at risk” couples often ask for prenatal diagnosis (PND). However, because loads of heteroplasmy (coexistence of mutant and wild type mtDNA) may vary among tissues and with time, the possibility that a single fetal sample may not reflect the whole neonate impedes prenatal diagnosis of mtDNA diseases. Methods: We carried out 13 prenatal diagnoses for the NARP (Neurogenic weakness, Ataxia, Retinitis Pigmentosa) m.8993T>G mtDNA mutation (p.Leu156Arg) in the ATP synthase subunit 6 gene. Analyses were performed on chorionic villous (CVS) and/or amniocytes (AS) samples carried out at various stages of pregnancy, using a method enabling quantification of low DNA amounts. Results: Maternal mutant loads ranged from 0 to 75% in blood and had no predictive value for the fetus status, except for women with no detectable mutant DNA, whose fetuses were constantly mutation free. In 8/13 PND, mutant load was below 30%. These children are healthy at 2 to 7 years of age. In 5/13 PND, mutant load ranged from 65 to 100% and parents preferred to terminate the pregnancies (15-22 weeks of gestation). Single-cell analysis of 20 trophoblastic cells and 21 amniocytes isolated from two affected fetuses found an average mutant load close to the overall CVS and AS mutant load, despite striking intercellular variation. m.8993T>G mutant loads, assessed in 7, 17, 11, and 5 different tissues from 4 abortions, were identical in all tissues from a given individual (mean 78±1.2%, 91±0.7%, 74±2%, and 63±1.6% for the 4 fetuses, respectively). Conclusions: Our results indicate that the placental/amniotic mutant loads do reflect the NARP mutant mtDNA load in the whole fetus, even when the sample amount is small, and suggest that heteroplasmy level remains stable during pregnancy, at least after 10 weeks of gestation. While these data establish the feasibility of PND for this mutation, assessing more precisely the correlation between mutant load and disease severity should further help interpreting PND results.

Objective: The aim of this study was to determine if there is a significant difference in the risk of developing Wilms tumour between patients with submicroscopic and those with visible deletions of the WT1 tumour suppressor gene. Methods: To determine which subjects had WT1 deletions, high-resolution chromosomal deletion analysis of the 11p13 region was carried out in 193 people with aniridia. The rationale for this was that aniridia is caused by loss of function of one copy of the PAX6 gene, and although most patients with aniridia have intragenic mutations, a proportion has deletions that also include the nearby WT1 gene. Fluorescence in situ hybridisation (FISH) analysis of patients with aniridia identifies people with WT1 deletions regardless of whether they have Wilms tumour, allowing the deletion size to be correlated with clinical outcome. Results: Wilms tumour was not observed in any case without a WT1 deletion. Of subjects in whom WT1 was deleted, 77% with submicroscopic deletions (detectable only by high-resolution FISH analysis) presented with Wilms tumour compared with 42.5% with visible deletions (detectable by microscopy). This difference was significant. Conclusions: High-resolution deletion analysis is a useful tool for assessing the risk of Wilms tumour in neonates with aniridia. People with submicroscopic WT1 deletions have a significantly increased risk of Wilms tumour, and a high level of vigilance should be maintained in such cases.

BACKGROUND: Benign familial neonatal seizures are most often caused by mutations in the voltage-gated potassium channel subunit gene KCNQ2. More than 60 mutations have been described in BFNS families, approximately half of which lead to protein truncation. The hypothesis of this study was that deletion or duplication of >or=1 exons of KCNQ2 could cause BFNS in cases without coding or splicing mutations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was used to test a group of 21 unrelated patients with clinical features consistent with either BFNS, benign familial neonatal-infantile seizures or sporadic neonatal seizures, for exonic deletions and duplications. RESULTS: Three deletions and one duplication mutation were identified in four familial cases and cascade testing of their available family members showed that the mutations segregated with the phenotype in each family. The junction fragment for one of the deletions was amplified by PCR and sequenced to characterise the breakpoint and verify that a deletion had occurred. CONCLUSIONS: Submicroscopic deletions or duplications of KCNQ2 are seen in a significant proportion of BFNS families: four of nine (44%) cases previously testing negative for coding or splice site mutation by sequencing KCNQ2 and KCNQ3. MLPA is an efficient second-tier testing strategy for KCNQ2 to identify pathogenic intragenic mutations not detectable by conventional DNA sequencing methods.

Background: The cell surface glycoprotein, E-Cadherin (CDH1) is a key regulator of adhesive properties in epithelial cells. Germline mutations in CDH1 are well-established as the defects underlying the Hereditary Diffuse Gastric Cancer (HDGC)syndrome: an increased risk of lobular breast cancer (LBC) has been described in HDGC kindreds. However, germline CDH1 mutations have not been described in LBC patients outside of HDGC families. We sought to investigate the frequency of germline CDH1 mutations in LBC patients with early onset disease or family histories of breast cancer without DGC. Methods: Germline DNA was analyzed in 23 women with invasive lobular or mixed ductal and lobular breast cancers who had at least one close relative with breast cancer or had themselves been diagnosed before age 45, had tested negative for a germline BRCA1 or BRCA2 mutation, and reported no personal or family history of diffuse gastric cancer. The full coding sequence of CDH1 including splice junctions was PCR amplified and screened for mutations using DHPLC and sequencing. Results: A novel germline CDH1 truncating mutation in the extracellular portion of the protein (517insA) was identified in one subject who had lobular breast cancer at age 42 and a first degree relative with invasive lobular breast cancer. Conclusions: Germline CDH1 mutations can be associated with invasive lobular breast cancer in the absence of diffuse gastric cancer. The finding, if confirmed, may have implications for management of individuals at risk for this breast cancer subtype, and compels clarification of the cancer risks in the syndrome.

l-2-hydroxyglutaric aciduria (l-2-HGA) is a neurometabolic disorder that produces a variety of clinical neurological deficits, including psychomotor retardation, seizures and ataxia. The biochemical hallmark of l-2-HGA is the accumulation of l-2-hydroxyglutaric acid (l-2-HG) in cerebrospinal fluid, plasma and urine. Mutations within the gene L2HGDH (Entrez Gene ID 79944) on chromosome 14q22 encoding L-2-hydroxyglutaric acid dehydrogenase have recently been shown to cause l-2-HGA in humans. Using a candidate gene approach in an outbred pet dog population segregating l-2-HGA, the causal molecular defect was identified in the canine homologue of L2HGDH and characterised. DNA sequencing and pedigree analysis indicate a common founder effect in the canine model. The canine model shares many of the clinical and MRI features of the disease in humans and represents a valuable resource as a spontaneous model of l-2-HGA.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS) is a congenital disorder characterised by growth retardation, facial dysmorphisms, skeletal abnormalities and mental retardation. Broad thumbs and halluces are the hallmarks of the syndrome. RSTS is associated with chromosomal rearrangements and mutations in the CREB-binding protein gene (CREBBP), also termed CBP, encoding the CREB-binding protein. Recently, it was shown that mutations in EP300, coding for the p300 protein, also cause RSTS. CBP and EP300 are highly homologous genes, which play important roles as global transcriptional coactivators. OBJECTIVE: To report the phenotype of the presently known patients with RSTS (n = 4) carrying germline mutations of EP300. RESULTS: The patients with EP300 mutations displayed the typical facial gestalt and malformation pattern compatible with the diagnosis of RSTS. However, three patients exhibited much milder skeletal findings on the hands and feet than typically observed in patients with RSTS. CONCLUSIONS: Part of the clinical variability in RSTS is explained by genetic heterogeneity. The diagnosis of RSTS must be expanded to include patients without broad thumbs or halluces

Background: During whole genome microarray-based comparative genomic hybridisation (array CGH) screening of subjects with idiopathic intellectual disability, we identified two unrelated individuals with a similar de novo interstitial microdeletion at 2p15-2p16.1. Both individuals share a similar clinical phenotype including moderate to severe intellectual disability, autism/autistic features, microcephaly, structural brain anomalies including cortical dysplasia/pachygyria, renal anomalies (multicystic kidney, hydronephrosis), digital camptodactyly, visual impairment, strabismus, neuromotor deficits, communication and attention impairments, and a distinctive pattern of craniofacial features. Dysmorphic craniofacial features include progressive microcephaly, flat occiput, widened inner canthal distance, small palpebral fissures, ptosis, long and straight eyelashes, broad and high nasal root extending to a widened, prominent nasal tip with elongated, smooth philtrum, rounding of the upper vermillion border and everted lower lips. Methods: Clinical assessments, and cytogenetic, array CGH and fluorescence in situ hybridisation (FISH) analyses were performed. Results: The microdeletions discovered in each individual measured 4.5 Mb and 5.7 Mb, spanning the chromosome 2p region from 57.2 to 61.7 Mb and from 56 to 61.7 Mb, respectively. Each deleted clone in this range demonstrated a dosage reduction from two to one copy in each proband except for clone RP11-79K21, which was present in three copies in each proband and in four copies in their respective parents (two per each chromosome 2 homologue). Discussion: The common constellation of features found in the two affected subjects indicates that they have a newly recognised microdeletion syndrome involving haploinsufficiency of one or more genes deleted within at least a 4.5-Mb segment of the 2p15-16.1 region.

The gap junction subunit connexin permits direct intercellular exchange of ions and molecules including glutamate, and plays an important role in the central nervous system. The connexin 40 (Cx40) and connexin 50 (Cx50) genes are located on chromosome 1q21.1, a region strongly linked with schizophrenia. These lines of evidence suggest that Cx40 and Cx50 may play a role in schizophrenia.Using an allele-specific PCR assay, four polymorphisms each were genotyped for Cx40 and Cx50 in 190 Caucasian patients with schizophrenia and 190 controls matched for sex, age and ethnicity. Following up, Cx50 rs989192 and rs4950495 were investigated in 99 Canadian and 163 Portuguese trios and nuclear families with schizophrenia probands. Hardy-Weinberg equilibrium and linkage disequilibrium (LD) block identification was carried out with HaploView, and association analysis for alleles and haplotypes with a permutation test of 10 000 simulations was carried out using the UNPHASED software program.Distributions of genotype frequencies of all markers were in Hardy-Weinberg equilibrium in Caucasian patients, controls and families. One rs989192-rs4950495 LD block was found in patients but not in controls. We found a significant association between the Cx50 rs989192-rs4950495 haplotype and schizophreniay (chi(2) = 29.55, p0.01). The A-C haplotype had a higher frequency in patients (chi(2) = 7.153, p0.01). Family studies also showed that the A-C haplotype was transmitted more often to patients with schizophrenia (chi(2) = 8.43, p0.01). No association of Cx40 with schizophrenia was found for allele, genotype or haplotype analyses.Our matched case-control and family study indicate that Cx50, but not Cx40, may play a role in the genetic susceptibility to schizophrenia.

Background: Autism is a common childhood neurodevelopmental disorder with suggested genetic background. About 5-10% of autism cases are associated with chromosomal abnormalities or monogenic disorders. However, the role of subtle genomic imbalances in autism has not been delineated. Here, we have tested a hypothesis suggesting autism to be associated with subtle genomic imbalances manifested as low-level chromosomal mosaicism. Methods: We surveyed stochastic (background) aneuploidy in children with/without autism by interphase three-colour fluorescence in situ hybridization (FISH). The rate of chromosome loss and gain involving six arbitrarily selected autosomes and sex chromosomes was assessed in peripheral blood cells of 60 unaffected children and 120 children with autism. Results: Four autistic boys from 120 analyzed (3.3%) with rare structural chromosomal abnormalities: 46,XY,t(1;6)(q42.1;q27); 46,XY,inv(2)(p11q13); 46,XY,der(6),ins(6;1)(q21;p13.3p22,1)pat; 46,XY,r(22)(p11q13) were excluded from further molecular cytogenetic analysis. Studying over 420,000 cells in 60 controls and 116 children with idiopathic autism, we have determined the mean frequency of stochastic aneuploidy in control and autism: (i) autosome loss - 0.58% (95% CI 0.42-0.75%) and 0.60% (95% CI 0.37-0.83%), respectively, p=0.83; (ii) autosome gain - 0.15% (95% CI 0.09-0.21%) and 0.22% (95% CI 0.14-0.30%), respectively, p=0.39; (iii) chromosome X gain - 1.11% (95% CI 0.90-1.31%) and 1.01% (95% CI 0.85-1.17%), respectively, p=0.30. The frequency of mosaic aneuploidy over the background level was found in 19 (16%) of 116 children with idiopathic autism, while outlier values were not found in controls (p=0.0019). Conclusions: Our findings identify low-level aneuploidy as a new genetic risk factor for autism. Therefore, molecular cytogenetic analysis of somatic mosaicism is warranted in children with unexplained autism.

Background: The Pierre Robin sequence (PRS), consisting of cleft palate, micrognathia and glossoptosis, can be seen as part of the phenotype in other Mendelian syndromes—for instance, campomelic dysplasia (CD) which is caused by SOX9 mutations—but the aetiology of non-syndromic PRS has not yet been unravelled. Objective: To gain more insight into the aetiology of PRS by studying patients with PRS using genetic and cytogenetic methods. Methods: 10 unrelated patients with PRS were investigated by chromosome analyses and bacterial artificial chromosome arrays. A balanced translocation was found in one patient, and the breakpoints were mapped with fluorescence in situ hybridisation and Southern blot analysis. All patients were screened for SOX9 and KCNJ2 mutations, and in five of the patients expression analysis of SOX9 and KCNJ2 was carried out by quantitative real-time PCR. Results: An abnormal balanced karyotype 46,XX, t(2;17)(q23.3;q24.3) was identified in one patient with PRS and the 17q breakpoint was mapped to 1.13 Mb upstream of the transcription factor SOX9 and 800 kb downstream of the gene KCNJ2 . Furthermore, a significantly reduced SOX9 and KCNJ2 mRNA expression was observed in patients with PRS. Conclusion: Our findings suggest that non-syndromic PRS may be caused by both SOX9 and KCNJ2 dysregulation.

Integrins are heterodimeric transmembrane glycoproteins that function as key adhesion and cell signalling receptors. A functional polymorphism in the integrin beta3 subunit encoded by the ITGB3 gene, Leu33Pro, has been shown to modify a variety of traits of beta3-expressing cells. To analyse the role of this functional polymorphism in modifying BRCA1-associated ovarian and breast cancer risks, a case-control study was performed among Polish BRCA1 mutation carriers including 319 breast cancer cases, 146 ovarian cancer cases and 290 controls unaffected by breast and ovarian cancer, in situ breast cancer or any other kind of cancer. Genotyping analysis was performed using PCR-based restriction fragment length polymorphism analysis. Odds ratios were calculated using univariate and multivariate logistic regression, taking into account a series of confounding variables, including the presence of related study subjects, that potentially could have biased any association. The results revealed that the ITGB3_Leu33Pro polymorphism was associated with a 2.5-fold increased risk of ovarian cancer, whereas no association with breast cancer risk was found. Thus, it appears that the ITGB3_Leu33Pro polymorphism may potentially increase the risk of ovarian cancer in Polish women with an inherited BRCA1 mutation.

International audience; BACKGROUND: Chromosomal rearrangements, arising from unequal recombination between repeated sequences, are found in a subset of patients with autism. Duplications involving loci associated with behavioural disturbances constitute an especially good candidate mechanism. The Williams-Beuren critical region (WBCR), located at 7q11.23, is commonly deleted in Williams-Beuren microdeletion syndrome (WBS). However, only four patients with a duplication of the WBCR have been reported to date: one with severe language delay and the three others with variable developmental, psychomotor and language delay. OBJECTIVE AND METHODS: In this study, we screened 206 patients with autism spectrum disorders for the WBCR duplication by quantitative microsatellite analysis and multiple ligation-dependent probe amplification. RESULTS: We identified one male patient with a de novo interstitial duplication of the entire WBCR of paternal origin. The patient had autistic disorder, severe language delay and mental retardation, with very mild dysmorphic features. CONCLUSION: We report the first patient with autistic disorder and a WBCR duplication. This observation indicates that the 7q11.23 duplication could be involved in complex clinical phenotypes, ranging from developmental or language delay to mental retardation and autism, and extends the phenotype initially reported. These findings also support the existence of one or several genes in 7q11.23 sensitive to gene dosage and involved in the development of language and social interaction.

A recent study, looking at the lifetime risk of developing malignant peripheral nerve sheath tumour (MPNST) in patients with neurofibromatosis type 1 (NF1), estimated the risk to be 8–13%. Prior to this, longitudinal studies had shown that patients with NF1 had a risk of 4–5% of developing MPNST, and cross‐sectional studies had found that only 1–2% of patients with NF1 had MPNST. The aim of this study was to estimate the lifetime risk of MPNST in patients with NF1 in southern Scotland, using patient records obtained from the Edinburgh and Glasgow Genetic Units and Scottish Cancer Register. In the period 1993–2002, 14 patients with NF1 were diagnosed with MPNST in a population of 3.5 million. The lifetime risk of MPNST in the Scottish patients with NF1 was calculated to be 5.9–10.3%. This provides further evidence that patients with NF1 are at greater risk of developing MPNST than was previously estimated, and emphasises the importance of educating patients about suspicious symptoms, which may need an urgent medical opinion. The mean age at diagnosis of MPNST (p

Background: Rare reports on patients with congenital myopathy with excess of muscle spindles (CMEMS), hypertrophic cardiomyopathy, and variable features resembling Noonan syndrome have been published, but the genetic basis of this condition has been unknown. Methods and Results: We analyzed PTPN11 and RAS genes in five unrelated patients with this phenotype and found HRAS mutations in four of them. Two disease-associated mutations, G12V and G12S, have previously been observed in patients with Costello syndrome (CS), while two other mutations, E63K and Q22K, are novel. All four mutations are predicted to enhance downstream H-Ras signaling, suggesting that CMEMS is a developmental consequence of sustained H-Ras activation in skeletal muscle. Conclusion: This type of myopathy may represent a previously unrecognized manifestation of CS. However, some patients carrying HRAS mutations may exhibit prominent congenital muscular dysfunction, while features of CS are less obvious, suggesting that germline HRAS mutations may underlie some cases of otherwise unclassified neonatal neuromuscular disorders.

A novel missense mutation in the mediator of RNA polymerase II transcription subunit 12 (MED12) gene has been found in the original family with Lujan syndrome and in a second family (K9359) that was initially considered to have Opitz–Kaveggia (FG) syndrome. A different missense mutation in the MED12 gene has been reported previously in the original family with FG syndrome and in five other families with compatible clinical findings. Neither sequence alteration has been found in over 1400 control X chromosomes. Lujan (Lujan–Fryns) syndrome is characterised by tall stature with asthenic habitus, macrocephaly, a tall narrow face, maxillary hypoplasia, a high narrow palate with dental crowding, a small or receding chin, long hands with hyperextensible digits, hypernasal speech, hypotonia, mild‐to‐moderate mental retardation, behavioural aberrations and dysgenesis of the corpus callosum. Although Lujan syndrome has not been previously considered to be in the differential diagnosis of FG syndrome, there are some overlapping clinical manifestations. Specifically, these are dysgenesis of the corpus callosum, macrocephaly/relative macrocephaly, a tall forehead, hypotonia, mental retardation and behavioural disturbances. Thus, it seems that these two X‐linked mental retardation syndromes are allelic, with mutations in the MED12 gene.

Contains fulltext : 52397.pdf (Publisher’s version ) (Closed access) BACKGROUND: Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. METHODS: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. RESULTS: Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. CONCLUSION: The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.

Facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant disorder, represents the third most common human muscular dystrophy. The FSHD disease locus, at chromosome 4q35, is associated with large contractions of the polymorphic repeat sequence array D4Z4. In addition to FSHD disease association with large D4Z4 deletions, a biased interaction with a specific 4qter subtelomeric sequence has been described in patients. Two distinct 4qter subtelomeres, defined as types 4qA and 4qB, have been identified and shown to be equally prevalent in the Caucasian population. In almost all 4q35‐linked patients with FSHD, however, disease expression only occurs when large D4Z4 deletions are located on 4qA‐defined 4qter subtelomeres. Conversely, large D4Z4 repeat contractions situated on 4qB‐defined subtelomeres either are not disease‐causing or exhibit a greatly reduced disease penetrance. This study was initiated to confirm this direct FSHD disease association data by measuring the frequency of type 4qA‐defined and 4qB‐defined subtelomeric sequences in a large cohort of 164 unrelated patients with FSHD from Turkey and the UK, all known to have large D4Z4 deletions. An almost complete association was found between large D4Z4 repeat array deletions located on 4qA‐defined 4qter subtelomeres and disease expression in our large FSHD patient cohort. The observed failure of probes 4qA and 4qB to hybridise to two patient‐derived DNA samples confirms the presence of an additional rare type of 4qter subtelomeric sequence in humans.

Codon reiteration disorders are caused by abnormal expansions of either polyglutamine or polyalanine tracts within the coding region of a protein. These mutations impair normal protein folding, resulting in aggregate formation in the affected tissues. Huntington's disease is the most common of the nine disorders caused by polyglutamine expansion mutations. The most extensively studied polyalanine expansion disorder is oculopharyngeal muscular dystrophy. There may be a link between diseases caused by polyglutamine and polyalanine expansion mutations as it has been shown that the expanded CAG/polyglutamine tract within the SCA3 gene can shift to the GCA[corrected]/polyalanine frame. Here, we show that this frameshifting phenomenon is more widespread and occurs in Huntington's disease. We have shown both +1 frameshift and +2 frameshift products (which may contain polyalanine or polyserine tracts, respectively) in human postmortem Huntington's disease brains and in a transgenic mouse model of Huntington's disease. Our data suggest that +1 and +2 frameshift products are generated at low levels. This may be relevant to the pathogenesis of human Huntington's disease, as we have previously shown that both polyserine and polyalanine-containing proteins are modifiers of mutant huntingtin toxicity, with low expression levels of polyalanine-containing proteins having a protective effect.

Background: X linked cone-rod dystrophy (CORDX) is a recessive retinal disease characterised by progressive dysfunction of photoreceptors. It is genetically heterogeneous, showing linkage to three X chromosomal loci. CORDX1 is caused by mutations in the RPGR gene (Xp21.1), CORDX2 is located on Xq27.2-28, and we recently localised CORDX3 to Xp11.4-q13.1. We aimed to identify the causative gene behind the CORDX3 phenotype. Methods: All 48 exons of the CACNA1F gene were screened for mutations by DNA sequencing. RNA from cultured lymphoblasts and peripheral blood activated T lymphocytes was analysed by RT-PCR and sequencing. Results: An ovelCACNA1F mutation, IVS28-1 GCGTC.TGG, in the splice acceptor site of intron 28 was identified. Messenger RNA studies indicated that the identified mutation leads to altered splicing of the CACNA1F transcript. Aberrant splice variants are predicted to result in premature termination and deletions of the encoded protein, Cav1.4 a1 subunit. Conclusion: CACNA1F mutations cause the retinal disorder, incomplete congenital stationary night blindness (CSNB2), although mutations have also been detected in patients with divergent diagnoses. Our results indicate that yet another phenotype, CORDX3, is caused by a mutation in CACNA1F. Clinically, CORDX3 shares some features with CSNB2 but is distinguishable from CSNB2 in that it is progressive, can begin in adulthood, has no nystagmus or hyperopic refraction, has only low grade astigmatism, and in dark adaptation lacks cone threshold and has small or no elevation of rod threshold. Considering all features, CORDX3 is more similar to other X chromosomal cone-rod dystrophies than to CSNB2.

Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem‐cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13–4 gene have recently been described in patients with FHL. We sequenced the Munc13–4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13–4 mutations were found, spread throughout the gene. Among novel mutations, 2650C→T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo‐immunotherapy was effective in all patients. Munc13–4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia.

Background: Mdm2 is a natural inhibitor of p53 function and its overexpression impairs p53 transcriptional activity. T→G single-nucleotide polymorphism at position 309 (SNP309) of mdm2 induces overexpression of mdm2, but inhibits p53. Objectives: To determine whether SNP309 is a risk-modifier polymorphism in colorectal cancer (CRC) and whether tumour selection of P53 mutations are influenced by SNP309. Methods: Single-stranded conformation polymorphism and automatic sequencing were performed. Results: SNP309 is not associated with the risk of CRC or recurrence of tumours. These data do not over-ride the tumour-selection capabilities of P53 mutations in CRC. However, a significant association with non-dominant-negative P53 mutations (p = 0.02) was found. Conclusions: MDM2 -SNP309 favours tumour selection of non-dominant negative P53 mutations in CRC, which also show an earlier age of tumour onset.

Craniosynostosis is a congenital developmental disorder involving premature fusion of cranial sutures, which results in an abnormal shape of the skull. Significant progress in understanding the molecular basis of this phenotype has been made for a small number of syndromic craniosynostosis forms. Nevertheless, in the majority of the ∼100 craniosynostosis syndromes and in non‐syndromic craniosynostosis the underlying gene defects and pathomechanisms are unknown. Here we report on a male infant presenting at birth with brachycephaly, proptosis, midfacial hypoplasia, and low set ears. Three dimensional cranial computer tomography showed fusion of the lambdoid sutures and distal part of the sagittal suture with a gaping anterior fontanelle. Mutations in the genes for FGFR2 and FGFR3 were excluded. Standard chromosome analysis revealed a de novo balanced translocation t(9;11)(q33;p15). The breakpoint on chromosome 11p15 disrupts the SOX6 gene, known to be involved in skeletal growth and differentiation processes. SOX6 mutation screening of another 104 craniosynostosis patients revealed one missense mutation leading to the exchange of a highly conserved amino acid (p.D68N) in a single patient and his reportedly healthy mother. The breakpoint on chromosome 9 is located in a region without any known or predicted genes but, interestingly, disrupts patches of evolutionarily highly conserved non‐genic sequences and may thus led to dysregulation of flanking genes on chromosome 9 or 11 involved in skull vault development. The present case is one of the very rare reports of an apparently balanced translocation in a patient with syndromic craniosynostosis, and reveals novel candidate genes for craniosynostoses and cranial suture formation.

Background: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is the autosomal dominant heritable syndrome with predisposition to development of renal cell carcinoma and smooth muscle tumours of the skin and uterus. Objective: To measure the fumarate hydratase (FH) enzyme activity in lymphoblastoid cell lines and fibroblast cell lines of individuals with HLRCC and other familial renal cancer syndromes. Methods: FH enzyme activity was determined in the whole cell, cytosolic, and mitochondrial fractions in 50 lymphoblastoid and 16 fibroblast cell lines including cell lines from individuals with HLRCC with 16 different mutations. Results: Lymphoblastoid cell lines (n = 20) and fibroblast cell lines (n = 11) from individuals with HLRCC had lower FH enzyme activity than cells from normal controls (p

Background: Idiopathic ( primary) hypertrophic cardiomyopathy (HCM) is mainly caused by mutations in genes encoding sarcomeric proteins. One of the most commonly mutated HCM genes is the myosin binding protein C (MYBPC3) gene. Mutations in this gene lead mainly to truncation of the protein which gives rise to a relatively mild phenotype. Pure HCM in neonates is rare and most of the time childhood HCM occurs in association with another underlying condition.Objective: To study the presence of mutations in the MYBPC3 gene in idiopathic childhood HCM.Methods: MYBPC3 coding region and splice junction variation were analysed by denaturing high performance liquid chromatography (DHPLC) and sequencing in DNA isolated from two neonates with severe unexplained HCM, who died within the first weeks of life.Results: Truncating mutations were found in both alleles of the MYBPC3 gene in both patients, suggesting there was no functional copy of the MYBPC3 protein. Patient 1 carried the maternally inherited c. 2373_2374insG mutation and the paternally inherited splice-donor site mutation c.1624+1G -> A. Patient 2 carried the maternally inherited frameshift mutation c.3288delA (p.Glu1096fsX92) and the paternally inherited non-sense mutation c.2827C -> T (p.Arg943X).Conclusions: The findings indicate the need for mutation analysis of genes encoding sarcomeric proteins in childhood HCM and the possibility of compound heterozygosity.

Contains fulltext : 50631.pdf (Publisher’s version ) (Closed access) BACKGROUND: Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA. OBJECTIVE: To analyse the sequences of the mtDNA coding region in 25 patients with OXPHOS system deficiency to identify the underlying genetic defect. RESULTS: Three novel non-synonymous substitutions in protein-coding genes, 4681T-->C in MT-ND2, 9891T-->C in MT-CO3 and 14122A-->G in MT-ND5, and one novel substitution in the 12S rRNA gene, 686A-->G, were found. The definitely pathogenic mutation 3460G-->A was identified in an 18-year-old woman who had severe isolated complex I deficiency and progressive myopathy. CONCLUSIONS: Bioinformatic analyses suggest a pathogenic role for the novel 4681T-->C substitution found in a boy with Leigh's disease. These results show that the clinical phenotype caused by the primary Leber's hereditary optic neuropathy mutation 3460G-->A is more variable than has been thought. In the remaining 23 patients, the role of mtDNA mutations as a cause of the OXPHOS system deficiency could be excluded. The deficiency in these children probably originates from mutations in the nuclear genes coding for respiratory enzyme subunits or assembly factors.

We report a novel 1 bp deletion (c.1834delC) in the MCT8 gene in a large Brazilian family with Allan‐Herndon‐Dudley syndrome (AHDS), an X linked condition characterised by severe mental retardation and neurological dysfunction. The c.1834delC segregates with the disease in this family and it was not present in 100 control chromosomes, further confirming its pathogenicity. This mutation causes a frameshift and the inclusion of 64 additional amino acids in the C‐terminal region of the protein. Pathogenic mutations in the MCT8 gene, which encodes a thyroid hormone transporter, results in elevated serum triiodothyronine (T3) levels, which were confirmed in four affected males of this family, while normal levels were found among obligate carriers. Through in vitro functional assays, we showed that this mutation decreases cellular T3 uptake and intracellular T3 metabolism. Therefore, the severe neurological defects present in the patients are due not only to deficiency of intracellular T3, but also to altered metabolism of T3 in central neurones. In addition, the severe muscle hypoplasia observed in most AHDS patients may be a consequence of high serum T3 levels.

Motivated by high throughput genotyping technology, our aim in this study was to experimentally compare the power and accuracy of case‐control and family trio based approaches for haplotype based, large scale, association gene mapping. We compared trio based and case‐control study designs in different disease models, and partitioned the performance differences into separate components: those from the sample ascertainment, the effective sample size, and the haplotyping approaches. For systematic and controlled tests, we simulated a rapidly expanding and relatively young isolated population. The experiments were also replicated with real asthma data. We used computationally efficient methods that scale up to large amounts of both markers and individuals. Mapping is based on a haplotype association test for haplotypes of 1–10 markers. For population based haplotype reconstruction, we use HaploRec, and compare it to both a simple trio based inference and true haplotypes. Firstly and surprisingly, statistically inferred population based haplotypes can be equally powerful as true haplotypes. Secondly, as expected, the effective sample size has a clear effect on both gene detection power and mapping accuracy. Thirdly, the sample ascertainment method does not have much effect on mapping accuracy. Finally, an interesting side result is that the simple haplotype association test clearly outperformed exhaustive allelic transmission disequilibrium tests. The results suggest that the case‐control design is a powerful alternative to the more laborious family based ascertainment approach, especially for large datasets, and wherever population stratification can be controlled.

Semaphorins are a large family of transmembrane proteins. The gene for SEMA4A encodes a transmembrane protein comprising 760 amino acids. To investigate its association with human retinal degeneration, mutation screening of the SEMA4A gene was carried out on 190 unrelated patients suffering from a variety of eye diseases. We report the first observation of the involvement of SEMA4A gene mutations causing retinitis pigmentosa (RP) and cone rod dystrophy (CRD). We screened the DNA of 135 patients with RP, 25 patients with CRD, and 30 with LCA using SSCP and direct DNA sequencing for mutations in the SEMA4A gene. Two mutations, p.D345H and p.F350C, were observed only in affected patients; they were not observed in any of the normal members or the 100 control subjects. Both mutations identified occur in the conserved semaphorin domain. Multiple sequence alignments using Clustal analysis showed that R713Q is a conserved substitution and D345H is a semi‐conserved substitution. We conclude that these mutations are a cause of various retinal degenerations.

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; p0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.

Background: Human autosomal recessive primary microcephaly (MCPH) is a heterogeneous disorder with at least six genetic loci (MCPH1–6), with MCPH5, caused by ASPM mutation, being the most common. Despite the high prevalence of epilepsy in microcephaly patients, microcephaly with frequent seizures has been excluded from the ascertainment of MCPH. Here, we report a pedigree with multiple affected individuals with microcephaly and seizures. Objective: To identify the gene responsible for microcephaly and seizures in this pedigree. Methods: Clinical assessments of three patients and brain MRIs of two patients were obtained. Genome-wide linkage screen with 10 k SNP microarray, fine mapping with microsatellite markers, and mutational analysis of the genomic DNA were performed on the pedigree. Results: We found that the family was linked to the MCPH5 locus on chromosome 1q31.2–q32.1. We screened ASPM and identified a previously unreported nonsense mutation that introduced a premature stop codon in exon 18 of the ASPM gene. Conclusions: We thus expand the clinical spectrum of ASPM mutations by showing that they can occur in patients with seizures and that the history of seizures alone should not necessarily preclude the diagnosis of primary microcephaly.

We have established the Developmental Genome Anatomy Project (DGAP; //dgap.harvard.edu) to take advantage of the unique opportunity to locate genes of developmental importance provided by apparently balanced chromosomal rearrangements associated with phenotypic abnormalities. By positional cloning at or near the breakpoints, we aim to identify the crucial disease genes whose functions have been disrupted by rearrangement.1 Kallmann’s syndrome (KS) is a developmental disorder characterised by anosmia resulting from agenesis of the olfactory lobes and hypogonadism secondary to deficiency of hypothalamic gonadotropin releasing hormone (GnRH). Its prevalence has been estimated at 1/10 000 in males and 1/50 000 in females. In a minority of cases there are inactivating mutations of KAL1, an X linked gene encoding a putative adhesion molecule thought to mediate embryonic neuronal migration.2,3 Constitutional autosomal chromosome translocations associated with KS have been reported, but the disrupted genes have not been identified.4–6 We have studied a white male subject with a de novo balanced translocation between chromosomes 7, in band p12.3, and 8, in band p11.2 (fig 1A), who was diagnosed on clinical examination to have hypogonadotropic hypogonadism (infantile testes), azoospermia, and cleft lip and palate, without frank anosmia. As a KS patient with a microdeletion involving the same 8p11.2 region had been reported, we sought to identify the chromosome 8 gene disrupted in this reciprocal translocation as a likely candidate for the cause of autosomal KS as well as of isolated hypogonadotropic hypogonadism.7 While this breakpoint in FGFR1 was being characterised, Dode et al identified FGFR1 mutations in several patients, establishing that disruption of FGFR1 can cause autosomal dominant KS.8 Figure 1 Fluorescent in situ hybridisation (FISH) mapping of the chromosome 8 breakpoint. (A) Ideogram illustrating the balanced t(7;8)(p12.3;p11.2) in the patient. (B) FISH mapping with RP11-100B16, labelled with SpectrumOrange, resulted in hybridisation …

Characteristic deformities of skull shape occur as a result of different patterns of sutural fusion, while compen- satory skull expansion occurs at unaffected sutures to accommodate the growing brain. Premature fusion of the sagittal suture, for example, results in anterior to posterior elongation of the skull known as scaphocephaly. 2 Sagittal synostosis is the most common type of craniosynostosis, occurring in 40-58% of cases reported in large neurosurgical surveys, and shows a male predominance. 3-6 Most of the cases in these reports are ''non-syndromic'' instances of sagittal synostosis, while familial cases represent 2-9% of the total. Mental retardation is uncommon in isolated sagittal synostosis, but is more common in cases with associated malformations. 4-6

Hearing impairment is a common and highly heterogeneous sensory disorder. Genetic causes are thought to be responsible for more than 60% of the cases in developed countries.1 In the majority of cases, non-syndromic hearing impairment is inherited in an autosomal recessive pattern.2 Thirty eight different loci and 20 genes for autosomal recessive non-syndromic hearing impairment (ARNSHI) have been identified to date.3 In many populations, up to 50% of all cases of ARNSHI are caused by mutations in the DFNB1 locus (MIM 220290) on 13q12.4 This locus contains the GJB2 gene (MIM 121011), encoding connexin-26 (Cx26),5 which belongs to a family of transmembrane proteins with about 20 members in humans. Hexamers of connexins (connexons) are displayed in the plasma membrane. Docking of connexons on the surfaces of two adjacent cells results in the formation of intercellular gap junction channels.6 Several different connexins, including Cx26, have been shown to participate in the complex gap junction networks of the cochlea.7,8 It has been postulated that these networks play a key role in potassium homeostasis, which is essential for the sound transduction mechanism.9 Given the high prevalence of DFNB1 deafness, molecular testing for GJB2 mutations has become the standard of care for the diagnosis of patients with non-syndromic hearing impairment of unknown cause.10 However, the finding of a large number of affected subjects with only one GJB2 mutant allele complicates the molecular diagnosis of DFNB1 deafness. In different studies, these have accounted for 10–50% of deaf subjects with GJB2 mutations.4 It was hypothesised that there could be other mutations in the DFNB1 locus but outside the GJB2 gene. This hypothesis gained support by the finding of a deletion in the DFNB1 locus outside GJB2 but truncating the neighbouring GJB6 gene (MIM 604418), which …

Hodgkin’s disease was recently designated Hodgkin lymphoma (HL) in the World Health Organization Classification.1 The National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) population based registries estimate that 7900 new cases are diagnosed annually in the USA.2 Clues to its aetiology have been suggested by the bimodal age distribution; higher risks in males, in people with higher socioeconomic status, and in smaller families; and occurrence of Epstein-Barr virus in HL tumour cells.3 The importance of genetic factors is indicated by reports of multiply affected families from case series,4–6 a twin study,7 a case–control study,8 and population registry studies carried out in Utah,9 Denmark,10 Israel,11 and Sweden.12–14 We recently analysed data from registries in Sweden and Denmark and found significant familial aggregation of HL and other lymphoproliferative tumours.15 The relative risk for HL among first degree relatives of cases compared with controls was 3.1. Relative risks were higher in males compared with females, and in siblings of cases compared with parents and offspring. Relatives of earlier onset cases were at higher risk for HL and for all lymphoproliferative tumours and were also at higher risk for developing early onset tumours themselves. These findings are consistent with those seen from earlier case series studies but have the advantage of being from large, population based samples. It is not known whether or how extrinsic risk factors interact with genetic susceptibility. Identifying inherited susceptibility genes is an important step towards defining the pathway(s) leading to development of HL and understanding its complex aetiology. There have been many studies of somatic mutations in HL tumour cells, but although there are associations with HLA types, specific germline genes causing susceptibility have not yet been identified. Early studies of HLA Class I alleles …

3-7 The extent of TNFa expression has been shown to be associated with the overall allele frequency and genotype distribution of the NcoI restriction fragment length polymorphism in the first intron of the TNFb locus. 89 In an in vitro endotoxin stimulation model, the extent of TNF expression has been correlated to the genotype of a single base polymorphism in the 2308 promoter region of the TNFa gene. 10 In contrast to this finding, the clinical importance of polymorphisms in the TNFa gene remains controversial. Survival in severe sepsis 11 has not been associated with genotype distribution of single base mutation in the 2308 promoter region of the TNFa gene. Focussing on neonatal morbidity, neither the development of chronic lung disease of prematurity 12 nor necrotising enterocolitis 13 were correlated to genotype distribution of the TNFa gene, whereas the development of intraventricular haemorrhage (IVH) was correlated to the 2308 promoter region polymorphism of the TNFa gene. 12 Genetic determina

Background: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. Objective and methods: Array-CGH with approximately 3500 large insert clones spaced at ∼1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. Results: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. Conclusions: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.

Background: Non-syndromic hearing loss is among the most genetically heterogeneous traits known in humans. To date, at least 50 loci for autosomal dominant non-syndromic sensorineural hearing loss (ADNSSHL) have been identified by linkage analysis. Objective: To report the mapping of a novel autosomal dominant deafness locus on the long arm of chromosome 14 at 14q11.2-q12, DFNA53, in a large multigenerational Chinese family with post-lingual, high frequency hearing loss that progresses to involve all frequencies. Results: A maximum multipoint LOD score of 5.4 was obtained for marker D14S1280. The analysis of recombinant haplotypes mapped DFNA53 to a 9.6 cM region interval between markers D14S581 and D14S1021. Four deafness loci (DFNA9, DFNA23, DFNB5, and DFNB35) have previously been mapped to the long arm of chromosome 14. The critical region for DFNA53 contains the gene for DFNA9 but does not overlap with the regions for DFNB5, DFNA23, or DFNB35. Screening of the COCH gene (DFNA9), BOCT , EFS , and HSPC156 within the DFNA53 interval did not identify the cause for deafness in this family. Conclusions: Identifying the DFNA53 locus is the first step in isolating the gene responsible for hearing loss in this large multigeneration Chinese family.

Background: Placental mesenchymal dysplasia (PMD) is a distinct syndrome of unknown aetiology that is associated with significant fetal morbidity and mortality. Intrauterine growth restriction is common, yet, paradoxically, many of the associated fetuses/newborns have been diagnosed with Beckwith-Wiedemann syndrome (BWS). Methods: We report two cases of PMD with high levels of androgenetic (complete paternal uniparental isodisomy) cells in the placenta and document, in one case, a likely androgenetic contribution to the fetus as well. Results: The same haploid paternal complement found in the androgenetic cells was present in coexisting biparental cells, suggesting origin from a single fertilisation event. Conclusions: Preferential allocation of the normal cells into the trophoblast explains the absence of trophoblast overgrowth, a key feature of this syndrome. Interestingly, the distribution of androgenetic cells appears to differ from that reported for artificially created androgenetic mouse chimeras. Androgenetic mosaicism for the first time provides an aetiology for PMD, and may be a novel mechanism for BWS and unexplained intrauterine growth restriction.

Background: Espins are actin bundling proteins present in hair cell stereocilia. A recessive mutation in the espin gene ( Espn ) has been detected in the jerker mouse and causes deafness, vestibular dysfunction, and hair cell degeneration. More recently mutations in the human espin gene ( ESPN ) have been described in two families affected by autosomal recessive hearing loss and vestibular areflexia. Objective: To report the identification of four additional ESPN mutations (S719R, D744N, R774Q, and delK848) in patients affected by autosomal dominant hearing loss without vestibular involvement. Results: To determine whether the mutated ESPN alleles affected the biological activity of the corresponding espin proteins in vivo, their ability to target and elongate the parallel actin bundles of brush border microvilli was investigated in transfected LLC-PK1-CL4 epithelial cells. For three mutated alleles clear abnormalities in microvillar length or distribution were obtained. Conclusions: The results further strengthen the causative role of the espin gene in non-syndromic hearing loss and add new insights into espin structure and function.

Background: A higher frequency of skewed X chromosome inactivation has been reported in a consecutive series of young patients with breast cancer compared with controls of a similar age. Objective: To investigate the X inactivation pattern in patients with familial non- BRCA1 / BRCA2 breast cancer (n = 272), BRCA1 / BRCA2 germline mutations (n = 35), and sporadic breast cancer (n = 292). Methods: X inactivation pattern was determined by polymerase chain reaction analysis of the highly polymorphic CAG repeat in the androgen receptor ( AR ) gene. The X inactivation pattern was classified as skewed when 90% or more of the cells preferentially expressed one X chromosome. Results: Young patients with familial breast cancer had a significantly higher frequency of skewed X inactivation (11.2%) than young controls (2.7%) (p = 0.001). There was also a strong tendency for middle aged patients with sporadic breast cancer to be more skewed than middle aged controls (13.6% v 4.4%) (p = 0.02). No association between skewed X inactivation and breast cancer was found for the BRCA1 / BRCA2 patients . Conclusions: Skewed X inactivation may be a risk factor for the development of breast cancer in both sporadic and familial breast cancer and may indicate an effect of X linked genes.

The CBP gene (OMIM #600140) encodes the 2441 amino acid cyclic AMP response element binding protein (CREB) binding protein (Mr 265 000) that functions as an essential co-activator for a number of transcription factors.1 In particular, CBP, like its homologue EP300, enhances gene transcription by linking sequence specific transcription factors to transcription factor IIB and the RNA polymerase II holoenzyme.2 CBP can also promote gene transcription by acetylation of histones through its histone acetyltransferase activity (HAT)3 and by acetylation of specific transcription factors such as p53.4–6 Several lines of evidence suggest the involvement of CBP in tumour formation. CBP interacts with the human T cell leukaemia virus (HTLV) Tax protein and v-myb to induce viral and cellular genes that promote cell transformation, while adenovirus E1A, simian virus SV40 Tag, and human papillomavirus (HPV) E6 bind to CBP and inhibit its co-activator function. CBP is also known to modulate the actions of the hMDM2 and AML1 cellular proto-oncogenes and the BRCA1 and p53 tumour suppressors.7 More direct evidence comes from genetic studies of translocation events involving CBP in leukaemia. In particular, the t(8;16)(p11;p13) translocation in AML involves a fusion of the CBP and MOZ genes,8 a novel t(10;16)(q22;p13) translocation in a childhood acute myelogenous leukaemia (AML-M5a) generates a MORF-CBP chimera,9 and the t(11;16)(q23;p13.3) translocation in topoisomerase II treated acute leukaemia or myelodysplasia results in a MLL-CBP fusion.10 In addition, haploinsufficiency of CBP in humans due to chromosomal rearrangements, microdeletions, and point mutations results in Rubenstein-Taybi Syndrome (RTS), and is associated with an increased risk for a variety of tumours.11 As a result of these observations, a number of studies have evaluated whether CBP is a direct mutational target in cancer and has tumour suppressor activity. One recent mutation screening study of …

Oral–facial–digital syndrome type 1 (OFD1) is characterised by an X linked dominant mode of inheritance with lethality in males. Clinical features include facial dysmorphism with oral, tooth, and distal abnormalities, polycystic kidney disease, and central nervous system malformations. Large interfamilial and intrafamilial clinical variability has been widely reported, and 18 distinct mutations have been previously reported within OFD1. A French and Belgian collaborative study collected 25 cases from 16 families. OFD1 was analysed using direct sequencing and phenotype–genotype correlation was performed using χ2 test. X inactivation studies were performed on blood lymphocytes. In 11 families, 11 novel mutations, including nine frameshift, one nonsense, and one missense mutation were identified, which spanned nine different exons. A combination of our results with previously reported cases showed that the majority of mutations (65.5%) was located in exons 3, 8, 9, 13, and 16. There was phenotype–genotype correlation between (a) polycystic kidney disease and splice mutations; (b) mental retardation and mutations located in exons 3, 8, 9, 13, and 16; and (c) tooth abnormalities and mutations located in coiled coil domains. Comparing the phenotype of the families with a pathogenic mutation to families with absence of OFD1 mutation, polycystic kidneys and short stature were significantly more frequent in the group with no OFD1 mutation, whereas lingual hamartomas were significantly more frequent in the group with OFD1 mutation. Finally, an X inactivation study showed non‐random X inactivation in a third of the samples. Differential X inactivation between mothers and daughters in two families with high intrafamilial variability was of particular interest. Slight phenotype–genotype correlations were established, and X inactivation study showed that skewed X inactivation could be partially involved in the pathogenesis of intrafamilial clinical variability.

Background: The vast majority of BRCA1 missense sequence variants remain uncharacterised for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. Objective: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. Methods and Results: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. Conclusions: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as “benign”. In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2 .