Rare structural variation of synapse and neurotransmission genes in autism.

Autism spectrum disorders (ASDs) comprise a constellation of highly heritable neuropsychiatric disorders. Genome-wide studies of autistic individuals have implicated numerous minor risk alleles but few common variants, suggesting a complex genetic model with many contributing loci. To assess commonality of biological function among rare risk alleles, we compared functional knowledge of genes overlapping inherited structural variants in idiopathic ASD subjects relative to healthy controls. In this study we show that biological processes associated with synapse function and neurotransmission are significantly enriched, with replication, in ASD subjects versus controls. Analysis of phenotypes observed for mouse models of copy-variant genes established significant and replicated enrichment of observable phenotypes consistent with ASD behaviors. Most functional terms retained significance after excluding previously reported ASD loci. These results implicate several new variants that involve synaptic function and glutamatergic signaling processes as important contributors of ASD pathophysiology and suggest a sizable pool of additional potential ASD risk loci.

Epidemiological investigation of a Legionnaires' disease outbreak in Christchurch, New Zealand: the value of spatial methods for practical public health.

Between April and August 2005 Christchurch, New Zealand experienced an outbreak of Legionnaires' disease. There were 19 laboratory-confirmed case including three deaths. Legionella pneumophila serogroup 1 (Lpsg1) was identified as the causative agent for all cases. A case-control study indicated a geographical association between the cases but no specific common exposures. Rapid spatial epidemiological investigation confirmed the association and identified seven spatially significant case clusters. The clusters were all sourced in the same area and exhibited a clear anisotropic process (noticeable direction) revealing a plume effect consistent with aerosol dispersion from a prevailing southwesterly wind. Four out of five cases tested had indistinguishable allele profiles that also matched environmental isolates from a water cooling tower within the centre of the clusters. This tower was considered the most probable source for these clusters. The conclusion would suggest a maximum dispersal distance in this outbreak of 11·6 km. This work illustrated the value of geostatistical techniques for infectious disease epidemiology and for providing timely information during outbreak investigations.

Perlecan, the major proteoglycan of basement membranes, is altered in patients with Schwartz-Jampel syndrome (chondrodystrophic myotonia).

Schwartz-Jampel syndrome (SJS1) is a rare autosomal recessive disorder characterized by permanent myotonia (prolonged failure of muscle relaxation) and skeletal dysplasia, resulting in reduced stature, kyphoscoliosis, bowing of the diaphyses and irregular epiphyses. Electromyographic investigations reveal repetitive muscle discharges, which may originate from both neurogenic and myogenic alterations. We previously localized the SJS1 locus to chromosome 1p34-p36.1 and found no evidence of genetic heterogeneity. Here we describe mutations, including missense and splicing mutations, of the gene encoding perlecan (HSPG2) in three SJS1 families. In so doing, we have identified the first human mutations in HSPG2, which underscore the importance of perlecan not only in maintaining cartilage integrity but also in regulating muscle excitability.

Changing epidemiological trends of legionellosis in New Zealand, 1979-2009.

This study evaluated the spatio-temporal variation of Legionella spp. in New Zealand using notification and laboratory surveillance data from 1979 to 2009 and analysed the epidemiological trends. To achieve this we focused on changing incidence rates and occurrence of different species over this time. We also examined whether demographic characteristics such as ethnicity may be related to incidence. The annual incidence rate for laboratory-proven cases was 2·5/100,000 and 1·4/100,000 for notified cases. Incidence was highest in the European population and showed large geographical variations between 21 District Health Boards. An important finding of this study is that the predominant Legionella species causing disease in New Zealand differs from that found in other developed countries, with about 30-50% of cases due to L. longbeachae and a similar percentage due to L. pneumophila for any given year. The environmental risk exposure was identified in 420 (52%) cases, of which 58% were attributed to contact with compost; travel was much less significant as a risk factor (6·5%). This suggests that legionellosis has a distinctive epidemiological pattern in New Zealand.

Rare structural variants found in attention-deficit hyperactivity disorder are preferentially associated with neurodevelopmental genes.

Attention-deficit/hyperactivity disorder (ADHD) is a common and highly heritable disorder, but specific genetic factors underlying risk remain elusive. To assess the role of structural variation in ADHD, we identified 222 inherited copy number variations (CNVs) within 335 ADHD patients and their parents that were not detected in 2026 unrelated healthy individuals. Although no excess CNVs, either deletions or duplications, were found in the ADHD cohort relative to controls, the inherited rare CNV-associated gene set was significantly enriched for genes reported as candidates in studies of autism, schizophrenia and Tourette syndrome, including A2BP1, AUTS2, CNTNAP2 and IMMP2L. The ADHD CNV gene set was also significantly enriched for genes known to be important for psychological and neurological functions, including learning, behavior, synaptic transmission and central nervous system development. Four independent deletions were located within the protein tyrosine phosphatase gene, PTPRD, recently implicated as a candidate gene for restless legs syndrome, which frequently presents with ADHD. A deletion within the glutamate receptor gene, GRM5, was found in an affected parent and all three affected offspring whose ADHD phenotypes closely resembled those of the GRM5 null mouse. Together, these results suggest that rare inherited structural variations play an important role in ADHD development and indicate a set of putative candidate genes for further study in the etiology of ADHD.

The impact of endonasal dacryocystorhinostomy (DCR), on patient health status as assessed by the Glasgow benefit inventory.

OBJECTIVE: To measure the effect of Endonasal DCR on the health status of patients, using a validated outcomes measure, the Glasgow Benefit Inventory. METHOD: Postal questionnaire with telephone follow up of patients undergoing Endonasal DCR in two institutions in Scotland. The same surgical technique is used in both centres. Patients were identified from prospectively collected data on consecutive patients undergoing this procedure. All adult patients, a minimum of twelve months post-intervention, were included. RESULTS: Ninety two of 123 patients (75%) completed the questionnaire, the mean age was 59 years and the sex ratio m:f was 1:1.8. The mean overall GBI for this intervention was + 32.7 (95% confidence intervals 27.8 - 37.6). The patients were grouped according to the indication for intervention: Obstruction of lacrimal system GBI + 32.7 (26.3-37.1), mucoecele + 40.1 (28.7-51.4), dacryocystitis + 19.4 (10.0-28.9). CONCLUSION: The GBI provides a measure of the effect of an ORL intervention on the health of a patient. Endonasal DCR scores highly when compared with a number of other rhinological procedures including rhinoplasty (GBI + 20), endoscopic sinus surgery (GBI + 23), and septal surgery (mean ranges from + 6 to + 24). Endonasal DCR is a successful intervention with demonstrable health benefits to the patient.

Measuring competence in endoscopic sinus surgery.

BACKGROUND: Competence based education is currently being introduced into higher surgical training in the UK. Valid and reliable performance assessment tools are essential to ensure competencies are achieved. No such tools have yet been reported in the UK literature. OBJECTIVE: We sought to develop and pilot test an Endoscopic Sinus Surgery Competence Assessment Tool (ESSCAT). The ESSCAT was designed for in-theatre assessment of higher surgical trainees in the UK. METHODS: The ESSCAT rating matrix was developed through task analysis of ESS procedures. All otolaryngology consultants and specialist registrars in Scotland were given the opportunity to contribute to its refinement. Two cycles of in-theatre testing were used to ensure utility and gather quantitative data on validity and reliability. Videos of trainees performing surgery were used in establishing inter-rater reliability. RESULTS: National consultation, the consensus derived minimum standard of performance, Cronbach's alpha = 0.89 and demonstration of trainee learning (p = 0.027) during the in vivo application of the ESSCAT suggest a high level of validity. Inter-rater reliability was moderate for competence decisions (Cohen's Kappa = 0.5) and good for total scores (Intra-Class Correlation Co-efficient = 0.63). Intra-rater reliability was good for both competence decisions (Kappa = 0.67) and total scores (Kendall's Tau-b = 0.73). CONCLUSION: The ESSCAT generates a valid and reliable assessment of trainees' in-theatre performance of endoscopic sinus surgery. In conjunction with ongoing evaluation of the instrument we recommend the use of the ESSCAT in higher specialist training in otolaryngology in the UK.

No CDKN2 mutations in neuroblastomas.

Mutations of CDKN2 have been found recently in melanoma and many other tumor types. Neuroblastoma shares with melanoma a neuroectodermal origin and a high incidence of deletions of the short arm of chromosome 1. Therefore, we analyzed 18 primary neuroblastomas and 9 tumor-derived cell lines for mutations in CDKN2. We used PCR-single-strand conformation polymorphism to examine exons 1 and 2 of the CDKN2 gene for mutations, but none were detected. Furthermore, no homozygous deletions were detected and there was no loss of heterozygosity at the closely linked IFNA locus. We conclude that disruption of the CDKN2 gene is not required for malignant transformation of human neuroblastomas.

Loss of heterozygosity for chromosomes 1 or 14 defines subsets of advanced neuroblastomas.

Neuroblastomas have been characterized genetically by N-myc amplification and by deletions or loss of heterozygosity (LOH) for the short arm of chromosome 1. However, recent studies have suggested deletion or allelic loss involving at least three other chromosome arms, 11q, 14q, and 17p. Therefore, we undertook an analysis of allelic loss for these respective chromosomal arms to determine the frequency and pattern of LOH as well as the correlation of these findings with other biological and clinical variables. A group of 24 pairs of normal and tumor DNAs was chosen that were representative of patients of different ages and stages. A substantial frequency of LOH (greater than or equal to 20%) was found only for 1p and 14q, whereas LOH for the other chromosome arms occurred in less than or equal to 5% of cases. On the basis of these results, we extended the analysis to a total of 59 neuroblastomas, and we found 1p LOH in 15 of the 59 cases (25%) and 14q LOH in 10 of 43 informative cases (23%). N-myc amplification was found in 15 of the 59 cases (25%). This analysis confirmed that 1p LOH and 14q LOH occurred almost exclusively in patients with advanced stages of disease. Furthermore, LOH for 1p and 14q usually occurred independent of each other, and 1p LOH frequently was associated with N-myc amplification, whereas 14q LOH was not. Thus, our results demonstrate that neuroblastomas are complex genetically and that there are at least two distinct loci for putative suppressor genes that are deleted independently in this tumor, both of which are associated with advanced stages of disease.

Homozygous deletion of CDKN2A (p16INK4a/p14ARF) but not within 1p36 or at other tumor suppressor loci in neuroblastoma.

Loss of heterozygosity of several specific genomic regions is frequently observed in neuroblastoma tumors and cell lines, but homozygous deletion (HD) is rare, and no neuroblastoma tumor suppressor gene (TSG) has yet been identified. We performed a systematic search for HD, indicative of a disrupted TSG, in a panel of 46 neuroblastoma cell lines. An initial search focused on a well-characterized consensus region of hemizygous deletion at 1p36.3, which occurs in 35% of primary neuroblastomas. Each cell line was screened with 162 1p36 markers, for a resolution of 13 kb within the consensus 1p36.3 deletion region and 350 kb throughout the remainder of 1p36. No HDs were detected. This approach was expanded to survey 21 known TSGs, specifically targeting intragenic regions frequently inactivated in other malignancies. HD was detected only at the CDKN2A (p16INK4a/p14ARF) gene at 9p21 and was observed in 4 of 46 cell lines. The observed region of HD included all exons of both CDKN2A and the closely linked CDKN2B (p15INK4b) gene for cell lines LA-N-6 and CHLA-174, all exons of CDKN2A but none of CDKN2B for CHLA-179, and only 104 bp within CDKN2A exon 2 for CHLA-101. All four deletions are predicted to inactivate the coding regions of both p16INK4a and p14ARF. HD was observed in corresponding primary tumor samples for CHLA-101 and CHLA-174 but was not present in constitutional samples. These results suggest that for neuroblastoma, large HDs do not occur within 1p36, most known TSGs are not homozygously deleted, and biallelic inactivation of CDKN2A may contribute to tumorigenicity in a subset of cases.

Consistent association of 1p loss of heterozygosity with pheochromocytomas from patients with multiple endocrine neoplasia type 2 syndromes.

Pheochromocytomas and medullary thyroid cancers (MTCs) are neuroendocrine tumors which arise sporadically or as part of the multiple endocrine neoplasia type 2 (MEN-2) hereditary syndromes. The most consistent molecular genetic abnormality which has been described in these tumors is loss of heterozygosity (LOH) of the short arm of chromosome 1 (1p). This finding is particularly interesting because the predisposition gene for the hereditary form of these tumors has been mapped to chromosome 10, but LOH on chromosome 10 in MEN-2 tumors is found rarely. We have used a battery of 1p DNA probes to elucidate the region of loss of 1p in 18 pheochromocytomas and 27 MTCs. Using restriction fragment length polymorphism analysis, we identified loss of all or a portion of 1p in 12 of 18 pheochromocytomas. 1p LOH was identified in nine of nine pheochromocytomas in MEN-2A and -2B patients, compared with only two of seven sporadic pheochromocytomas. We also found 1p LOH in one of two von Hippel-Lindau patients. LOH on 1p was noted in only three of 24 informative MTCs, and these were from patients with MEN-2A. In most of the pheochromocytomas, the entire short arm of chromosome 1p appears to have been lost; however, in three of the non-MEN pheochromocytomas and in three MEN-2A MTCs, the region of loss is smaller, allowing estimation of the smallest region of overlap. The combined data for MTCs and pheochromocytomas suggest that the smallest region of overlap of LOH is bounded by D1S15 (1pter-p22) and D1Z2 (1P36.3), excluding a region around MYCL (1p32). Although other regions of 1p should not be completely ruled out, the data suggest that this region may harbor a tumor suppressor gene or genes whose inactivation is important in the development of these tumors. Furthermore, the strong association between 1p LOH and the MEN-2 syndromes, especially in pheochromocytomas, suggests a relationship between the predisposition gene on chromosome 10 and the loss of the suppressor gene on 1p. Alternatively, other loci may be more important in sporadic disease.

Significance of chromosome 1p loss of heterozygosity in neuroblastoma.

We analyzed 156 primary neuroblastoma tumor samples for loss of heterozygosity at the distal short arm of chromosome 1 (1p LOH). We also compared 1p LOH with known clinical and genetic prognostic variables as well as patient outcome. 1p LOH was detected in 30 of 156 tumors (19%) and was strongly associated with adverse clinical and biological features. 1p LOH was also strongly predictive of a poor outcome in univariate analyses (estimated 4-year survival, 32 +/- 10% SE versus 76 +/- 5% SE; P < 0.001). However, the prognostic value of 1p LOH was equivocal when stratified for amplification of the MYCN oncogene (P = 0.16). We conclude that 1p LOH is an important component of a pattern of genetic abnormalities in neuroblastoma associated with an aggressive clinical course.

Identification of a consistent region of allelic loss on 1p32 in meningiomas: correlation with increased morbidity.

Meningioma is a common tumor of the central nervous system. Deletions of the short arm of chromosome 1 (1p) are the second most commonly observed chromosomal abnormality in these tumors. Here, we analyzed tumor and normal DNAs from 157 meningioma patients using PCR-based polymorphic loci. Loss of heterozygosity (LOH) for at least one informative marker on 1p was observed in 54 cases (34%), whereas LOH on 1q occurred in only 9 cases (8%). High-resolution deletion mapping defined a consensus region of deletion flanked distally by D1S2713 and proximally by D1S2134, which spans 1.5 cM within 1p32. LOH in this region has also been observed in several other malignancies, suggesting the presence of a tumor suppressor gene or genes that are important for several types of cancer. Statistical analysis revealed that 1p LOH was associated with chromosome 22 deletions and with abnormalities of the NF2 gene in meningioma. In addition, unlike other clinical and molecular characteristics, only 1p LOH was shown to be significantly associated with recurrence-free survival.

Familial predisposition to neuroblastoma does not map to chromosome band 1p36.

Familial predisposition to neuroblastoma, a common embryonal cancer of childhood, segregates as an autosomal dominant trait with high penetrance. It is therefore likely that neuroblastoma susceptibility is due to germ line mutations in a tumor suppressor gene. Cytogenetic, functional, and molecular studies have implicated chromosome band 1p36 as the most likely region to contain a suppressor gene involved in sporadic neuroblastoma tumorigenesis. We now demonstrate that neuroblastoma predisposition does not map to any of eight polymorphic markers spanning 1p36 by linkage analysis in three families. In addition, there is no loss of heterozygosity at any of these markers in tumors from affected members of these kindreds. Furthermore, there is strong evidence against linkage to two Hirschsprung disease (a condition that can cosegregate with neuroblastoma) susceptibility genes, RET and EDNRB. We conclude that the neuroblastoma susceptibility gene is distinct from the 1p36 tumor suppressor and the currently identified Hirschsprung disease susceptibility genes.

Physical mapping of the DDX1 gene to 340 kb 5' of MYCN.

One of the most important prognostic factors in neuroblastoma is amplification of the MYCN gene, which is strongly associated with advanced stages of disease and a poor prognosis. Although the MYCN amplicon sometimes spans more than 1 Mb, no other consistently expressed sequences from the MYCN amplicon have been reported. However, DDX1, a gene encoding a DEAD box protein, was recently mapped to chromosome 2p24 and is frequently co-amplified with MYCN. Therefore, we performed genomic mapping with YACs to determine the physical relationship between DDX1 and MYCN, and whether DDX1 was contained within the core region of amplification. Based on YAC restriction mapping and content analysis, DDX1 maps 340 kb 5' of MYCN, outside the core domain of consistent amplification. Interestingly, we also determined by sequence analysis and detailed restriction mapping that G21, previously isolated as a 'neuroblastoma-specific' cDNA clone from an MYCN amplicon, is a partial cDNA of DDX1. Our data confirm that DDX1 is amplified in some but not all MYCN-amplified tumors, and that it is rearranged in other cases. This suggests that the co-amplification of DDX1 is due to its proximity to MYCN.

Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas.

Deletions of the long arm of chromosome 11 (11q) have been noted in primary neuroblastomas, but a comprehensive analysis has not been performed. Therefore, we analysed 331 neuroblastomas (295 sporadic, 15 familial and 21 tumor-derived cell lines) to determine the prevalence of 11q allelic deletions, to map the location of a putative tumor suppressor gene and to perform clinical correlative studies. Assays for loss of heterozygosity (LOH) were performed at 24 microsatellite loci spanning 11q. LOH was observed at multiple 11q loci in 129/295 (44%) sporadic neuroblastomas, 5/15 (33%) familial neuroblastomas, and 5/21 (24%) neuroblastoma cell lines. A single region of 2.1 cM within 11q23.3, flanked by markers D11S1340 and D11S1299, was deleted in all specimens with 11q LOH. Allelic loss at 11q23 was inversely related to MYCN amplification (P<0.001). Within the subset of cases with a single copy of MYCN, 11q LOH was associated with advanced stage disease (P=0.008), unfavorable histopathology (P=0.042), and decreased overall survival probability (P=0.008). However, 11q LOH was not independently prognostic in multivariate analyses. These data support the hypothesis that a tumor suppressor gene mapping within 11q23.3 is commonly inactivated during the malignant evolution of a large subset of neuroblastomas, especially those with unamplified MYCN.

Loss of heterozygosity at 1p36 independently predicts for disease progression but not decreased overall survival probability in neuroblastoma patients: a Children's Cancer Group study.

PURPOSE: To determine the independent prognostic significance of 1p36 loss of heterozygosity (LOH) in a representative group of neuroblastoma patients. PATIENTS AND METHODS: Diagnostic tumor specimens from 238 patients registered onto the most recent Children's Cancer Group phase III clinical trials were assayed for LOH with 13 microsatellite polymorphic markers spanning chromosome band 1p36. Allelic status at 1p36 was correlated with other prognostic variables and disease outcome. RESULTS: LOH at 1p36 was detected in 83 (35%) of 238 neuroblastomas. There was a correlation of 1p36 LOH with age at diagnosis greater than 1 year (P = .026), metastatic disease (P<.001), elevated serum ferritin level (P<.001), unfavorable histopathology (P<.001), and MYCN oncogene amplification (P<.001). LOH at 1p36 was associated with decreased event-free survival (EFS) and overall survival (OS) probabilities (P<.0001). For the 180 cases with single-copy MYCN, 1p36 LOH status was highly correlated with decreased EFS (P = .0002) but not OS (P = .1212). Entering 1p36 LOH into a multivariate regression model suggested a trend toward an independent association with decreased EFS (P = .0558) but not with decreased OS (P = .3687). Furthermore, allelic status at 1p36 was the only prognostic variable that was significantly associated with decreased EFS in low-risk neuroblastoma patients (P = .0148). CONCLUSION: LOH at 1p36 is independently associated with decreased EFS, but not OS, in neuroblastoma patients. Determination of 1p36 allelic status may be useful for predicting which neuroblastoma patients with otherwise favorable clinical and biologic features are more likely to have disease progression.

Gene amplification in PNETs/medulloblastomas: mapping of a novel amplified gene within the MYCN amplicon.

OBJECTIVES: The pathological entity of primitive neuroectodermal tumour/medulloblastoma (PNET/MB) comprises a very heterogeneous group of neoplasms on a clinical as well as on a molecular level. We evaluated the importance of DNA amplification in medulloblastomas and other primitive neuroectodermal tumours (PNETs) of the CNS. METHOD: Restriction landmark genomic scanning (RLGS), a method that allows the detection of low level amplification, was used. RLGS provides direct access to DNA sequences circumventing positional cloning efforts. Furthermore, we analysed several samples by CGH. DESIGN: Twenty primary medulloblastomas, five supratentorial PNETs, and five medulloblastoma cell lines were studied. RESULTS: Although our analysis confirms that gene amplification is generally a rare event in childhood PNET/MB, we found a total of 17 DNA fragments that were amplified in seven different tumours. Cloning and sequencing of several of these fragments confirmed the previous finding of MYC amplification in the cell line D341 Med and identified novel DNA sequences amplified in PNET/MB. We describe for the first time amplification of the novel gene, NAG, in a subset of PNET/MB. Despite genomic amplification, NAG was not overexpressed in the tumours studied. We have determined that NAG maps less than 50 kb 5' of DDX1 and approximately 400 kb telomeric of MYCN on chromosome 2p24. CONCLUSION: We found a similar but slightly higher frequency of amplification than previously reported. We present several DNA fragments that may belong to the CpG islands of novel genes amplified in a small subset of PNET/MB. As an example we describe for the first time the amplification of NAG in the MYCN amplicon in PNET/MB.