Recommendations of the 2006 Human Variome Project meeting.

Lists of variations in genomic DNA and their effects have been kept for some time and have been used in diagnostics and research. Although these lists have been carefully gathered and curated, there has been little standardization and coordination, complicating their use. Given the myriad possible variations in the estimated 24,000 genes in the human genome, it would be useful to have standard criteria for databases of variation. Incomplete collection and ascertainment of variants demonstrates a need for a universally accessible system. These and other problems led to the World Heath Organization-cosponsored meeting on June 20-23, 2006 in Melbourne, Australia, which launched the Human Variome Project. This meeting addressed all areas of human genetics relevant to collection of information on variation and its effects. Members of each of eight sessions (the clinic and phenotype, the diagnostic laboratory, the research laboratory, curation and collection, informatics, relevance to the emerging world, integration and federation and funding and sustainability) developed a number of recommendations that were then organized into a total of 96 recommendations to act as a foundation for future work worldwide. Here we summarize the background of the project, the meeting and its recommendations.

Reinforcing the association between distal 1q CNVs and structural brain disorder: A case of a complex 1q43-q44 CNV and a review of the literature.

Copy Number Variations (CNVs) comprising the distal 1q region 1q43-q44 are associated with neurological impairments, structural brain disorder, and intellectual disability. Here, we report an extremely rare, de novo case of a 1q43-q44 deletion with an adjacent duplication, associated with severe seizures, microcephaly, agenesis of the corpus callosum, and pachygyria, a consequence of defective neuronal migration disorder. We conducted a literature survey to find that our patient is only the second case of such a 1q43-q44 CNV ever to be described. Our data support an association between 1q43-q44 deletions and microcephaly, as well as an association between 1q43-q44 duplications and macrocephaly. We compare and contrast our findings with previous studies reporting on critical 1q43-q44 regions and their constituent genes associated with seizures, microcephaly, and corpus callosum abnormalities [Ballif et al., 2012; Hum Genet 131:145-156; Nagamani et al., 2012; Eur J Hum Genet 20:176-179]. Taken together, our study reinforces the association between 1q43-q44 CNVs and brain disorder.

Development and validation of a family history screening questionnaire in Australian primary care.

PURPOSE: We aimed to validate a family history screening questionnaire in an Australian primary care population designed to identify people at increased risk for breast, ovarian, colorectal, and prostate cancer; melanoma; ischemic heart disease; and type 2 diabetes. METHODS: We prospectively validated the questionnaire in 6 general practices in Perth, Western Australia among 526 patients aged 20 to 50 years who responded to a single invitation from their general practice. They completed the 15-item questionnaire before a reference standard 3-generation pedigree was obtained by a genetic counselor blinded to the questionnaire responses. We calculated diagnostic performance statistics for the questionnaire using the pedigree as the reference standard. RESULTS: A combination of 9 questions had the following diagnostic performance, expressed as value (95% CI), to identify increased risk of any of the 7 conditions: area under the receiver operating characteristic curve 84.6% (81.2%-88.1%), 95% sensitivity (92%-98%), and 54% specificity (48%-60%). The combination of questions to detect increased risk had sensitivity of 92% (84%-99%) and 96% (93%-99%) for the 5 and 6 conditions applicable only to men and women, respectively. The specificity was 63% (28%-52%) for men and 49% (42%-56%) for women. The positive predictive values were 67% (56%-78%) and 68% (63%-73%), and the false-positive rates were 9% (0.5%-17%) and 9% (3%-15%) for men and women, respectively. CONCLUSIONS: This simple family history screening questionnaire shows good performance for identifying primary care patients at increased disease risk because of their family history. It could be used in primary care as part of a systematic approach to tailored disease prevention.

Linking MECP2 and pain sensitivity: the example of Rett syndrome.

Recent animal studies suggest links between MeCP2 function and sensitivity to pain. This study investigated the nature and prevalence of atypical pain responses in Rett syndrome and their relationships with specific MECP2 mutations. Families enrolled in the Australian Rett Syndrome Database (ARSD) and InterRett database participated in this study. Cases with a known MECP2 pathogenic mutation, whose families had completed a questionnaire on registration and had answered questions on pain sensitivity were included (n = 646). Logistic regression was used to analyze relationships between the atypical pain responses and genotype. Descriptions of decreased pain sensitivity were content analyzed. The prevalence estimate of reporting an abnormal pain response was 75.2% and a decreased sensitivity to pain was 65.0% in the population-based ARSD. Families of ARSD and InterRett subjects with a C-terminal (OR 2.6; 95% CI 0.8-8.0), p.R168X (OR 2.1; 95% CI 0.7-6.1), or p.R306C (OR 2.7; 95% CI 0.8-9.6) mutation were more likely to report decreased sensitivity to pain. Parents and carers described decreased and delayed responses in situations judged likely to cause pain such as injections, falls, trauma, and burns. This study has provided the first precise estimate of the prevalence of abnormal sensitivity to pain in Rett syndrome but specific relationships with genotype are not yet clear. Clinical practice should include a low threshold for the clinical assessment of potential injuries in Rett syndrome.

MECP2 genomic structure and function: insights from ENCODE.

MECP2, a relatively small gene located in the human X chromosome, was initially described with three exons transcribing RNA from which the protein MeCP2 was translated. It is now known to have four exons from which two isoforms are translated; however, there is also evidence of additional functional genomic structures within MECP2, including exons potentially transcribing non-coding RNAs. Accompanying the recognition of a higher level of intricacy within MECP2 has been a recent surge of knowledge about the structure and function of human genes more generally, to the extent that the definition of a gene is being revisited. It is timely now to review the published and novel functional elements within MECP2, which is proving to have a complexity far greater than was previously thought.

Unified criteria for ultrasonographic diagnosis of ADPKD.

Individuals who are at risk for autosomal dominant polycystic kidney disease are often screened by ultrasound using diagnostic criteria derived from individuals with mutations in PKD1. Families with mutations in PKD2 typically have less severe disease, suggesting a potential need for different diagnostic criteria. In this study, 577 and 371 at-risk individuals from 58 PKD1 and 39 PKD2 families, respectively, were assessed by renal ultrasound and molecular genotyping. Using sensitivity data derived from genetically affected individuals and specificity data derived from genetically unaffected individuals, various diagnostic criteria were compared. In addition, data sets were created to simulate the PKD1 and PKD2 case mix expected in practice to evaluate the performance of diagnostic criteria for families of unknown genotype. The diagnostic criteria currently in use performed suboptimally for individuals with mutations in PKD2 as a result of reduced test sensitivity. In families of unknown genotype, the presence of three or more (unilateral or bilateral) renal cysts is sufficient for establishing the diagnosis in individuals aged 15 to 39 y, two or more cysts in each kidney is sufficient for individuals aged 40 to 59 y, and four or more cysts in each kidney is required for individuals > or = 60 yr. Conversely, fewer than two renal cysts in at-risk individuals aged > or = 40 yr is sufficient to exclude the disease. These unified diagnostic criteria will be useful for testing individuals who are at risk for autosomal dominant polycystic kidney disease in the usual clinical setting in which molecular genotyping is seldom performed.

Correlation between clinical severity in patients with Rett syndrome with a p.R168X or p.T158M MECP2 mutation, and the direction and degree of skewing of X-chromosome inactivation.

INTRODUCTION: Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder that is usually associated with mutations in the MECP2 gene. The most common mutations in the gene are p.R168X and p.T158M. The influence of X-chromosome inactivation (XCI) on clinical severity in patients with RTT with these mutations was investigated, taking into account the extent and direction of skewing. METHODS: Female patients and their parents were recruited from the UK and Australia. Clinical severity was measured by the Pineda Severity and Kerr profile scores. The degree of XCI and its direction relative to the X chromosome parent of origin were measured in DNA prepared from peripheral blood leucocytes, and allele-specific polymerase chain reaction was used to determine the parental origin of mutation. Combining these, the percentage of cells expected to express the mutant allele was calculated. RESULTS: Linear regression analysis was undertaken for fully informative cases with p.R168X (n = 23) and p.T158M (n = 20) mutations. A statistically significant increase in clinical severity with increase in the proportion of active mutated allele was shown for both the p.R168X and p.T158M mutations. CONCLUSIONS: XCI may vary in neurological and haematological tissues. However, these data are the first to show a relationship between the degree and direction of XCI in leucocytes and clinical severity in RTT, although the clinical utility of this in giving a prognosis for individual patients is unclear.

A review of structural brain abnormalities in Pallister-Killian syndrome.

BACKGROUND: Pallister-Killian syndrome (PKS) is a rare multisystem developmental syndrome usually caused by mosaic tetrasomy of chromosome 12p that is known to be associated with neurological defects. METHODS: We describe two patients with PKS, one of whom has bilateral perisylvian polymicrogyria (PMG), the other with macrocephaly, enlarged lateral ventricles and hypogenesis of the corpus callosum. We have also summarized the current literature describing brain abnormalities in PKS. RESULTS: We reviewed available cases with intracranial scans (n = 93) and found a strong association between PKS and structural brain abnormalities (77.41%; 72/93). Notably, ventricular abnormalities (45.83%; 33/72), abnormalities of the corpus callosum (25.00%; 18/72) and cerebral atrophy (29.17%; 21/72) were the most frequently reported, while macrocephaly (12.5%; 9/72) and PMG (4.17%; 3/72) were less frequent. To further understand how 12p genes might be relevant to brain development, we identified 63 genes which are enriched in the nervous system. These genes display distinct temporal as well as region-specific expression in the brain, suggesting specific roles in neurodevelopment and disease. Finally, we utilized these data to define minimal critical regions on 12p and their constituent genes associated with atrophy, abnormalities of the corpus callosum, and macrocephaly in PKS. CONCLUSION: Our study reinforces the association between brain abnormalities and PKS, and documents a diverse neurogenetic basis for structural brain abnormalities and impaired function in children diagnosed with this rare disorder.

PKDB: Polycystic Kidney Disease Mutation Database--a gene variant database for autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) arises from mutations in the PKD1 and PKD2 genes. The Polycystic Kidney Disease Mutation Database (PKDB) is an internet-accessible relational database containing comprehensive information about germline and somatic disease-causing variants within these two genes, as well as polymorphisms and variants of indeterminate pathogenicity. The PKDB database structure incorporates an interface between these gene variant data and any associated patient clinical data. An initiative of the Polycystic Kidney Disease Foundation, PKDB is a publicly accessible database that aims to streamline the evaluation of PKD1 and PKD2 gene variants detected in samples from those with ADPKD, as well as to assist ongoing clinical and molecular research in the field. As the accurate reporting of nucleotide variants is essential for ensuring the quality of data within PKDB, a mutation checker has been mounted on the PKDB server allowing contributors to assess the accuracy of their PKD1 and PKD2 variant reports. Researchers and clinicians may submit their PKD1/PKD2 gene variants and any associated deidentified clinical data via standardized downloadable data entry forms accessible through the PKDB site. PKDB has been launched with the full details of PKD1 and PKD2 gene variant reports published in 73 peer-reviewed articles. Through a series of user-friendly advanced search facilities, users are able to query the database as required. The PKDB server is accessible at http://pkdb.mayo.edu.

Seizures in Rett syndrome: an overview from a one-year calendar study.

BACKGROUND: Rett syndrome is a neurodevelopmental disorder mainly affecting females. It is principally caused by mutations in the MECP2 gene. Seizures occur in about 80% of subjects but there has been little research into the factors contributing to their frequency. AIMS: To investigate seizure frequency in Rett syndrome and its relationship with other factors, including genetic characteristics and the use of anti-epileptic drugs. METHODS: Information on daily seizure occurrence and health service utilization and monthly anti-epileptic drug use was provided on 162 Rett syndrome cases for a calendar year. Age at onset of seizures, developmental history and other clinical and genetic characteristics were obtained from a contemporaneously completed questionnaire and from the Australian Rett Syndrome Database. Negative binomial regression was used to investigate factors associated with seizure rates. RESULTS: Seizure rates were highest in the 7-12 year age group. They were lower in those with p.R294X, p.R255X mutations and C terminal mutations. Those who had early developmental problems and poorer mobility had higher seizure rates as did those with greater clinical severity and poorer functional ability. Many different combinations of medications were being used with carbamazepine, sodium valproate and lamotrigine either singly or in combination with another being the most common. CONCLUSIONS: Seizure frequency in Rett syndrome is age-dependent, more common in those with more severe early developmental problems and influenced by mutation type.

Cytokine levels and associations with symptom severity in male and female children with autism spectrum disorder.

Background: Autism spectrum disorders (ASDs) are complex, pervasive, and heterogeneous neurodevelopmental conditions with varying trajectories, significant male bias and largely unknown etiology. However, an understanding of the biological mechanisms driving pathophysiology is evolving. Immune system aberrations, as identified through cytokine profiles, are believed to have a role in ASD. Altered cytokine levels may facilitate identification of ASD subtypes as well as provide biological markers of response to effective treatments. Research exploring the relationship between cytokine profiles and ASD symptoms is, however, in its infancy. The objective of this study was to explore relationships between cytokine levels and the severity of ASD and other clinical traits. Methods: Multiplex assay techniques were used to measure levels of 27 cytokines in plasma samples from a cohort of 144 children diagnosed with ASD. Results: Overall, results showed a significant negative association between platelet-derived growth factor (PDGF)-BB, and the severity of ASD symptoms. Furthermore, a significant interaction with sex suggested a different immune profile for females compared to males. ASD symptom severity was negatively associated with levels of 4 cytokines, IL-1ß, IL-8, MIP-1ß, and VEGF, in females, but not in males. Conclusions: Results of the present study suggest that an altered cytokine response or profile is associated with the severity of ASD-related symptoms, with sex a potential modifier of this relationship. Further research in larger populations which recognizes the importance of sex comparisons and longitudinal assessments are now required to extend and further describe the role of the immune system in ASD.

p.R270X MECP2 mutation and mortality in Rett syndrome.

Among cases in the Australian Rett Syndrome Database, the nonsense mutation p.R270X is one of the most commonly occurring single pathogenic MECP2 mutations. In two recent published reports of the MECP2 mutational spectrum the p.R270X appeared to be under represented. We hypothesised that increased mortality arising from this mutation may underlie this apparent discrepancy. We investigated our hypothesis in two independent study groups from Australia and the UK with prospective data collections (total n=524). Only females with Rett syndrome and an identified MECP2 mutation were included. Significant differences in survival were detected among Rett syndrome cases grouped for the eight most frequent mutations (log-rank chi(2) (7)=15.71, P=0.03). Moreover, survival among cases with p.R270X, when compared with survival among cases with all the other mutations was reduced (log-rank chi(2) (2)=6.94, P=0.01). Our observation of a reduced survival associated with the p.R270X mutation offers an explanation for the under representation of p.R270X in older subjects with Rett syndrome.

Screening for hereditary haemochromatosis within families and beyond.

Screening programmes for haemochromatosis that include follow-up identification of relatives are claimed to be cost effective. We assessed uptake of screening by first-degree relatives of two groups of index cases: people homozygous for the C282Y mutation ascertained by genetic screening of blood donors; and patients presenting clinically with haemochro matosis. Only 40 (24%) of 165 relatives of blood donors had been tested. By contrast, testing uptake in 121 relatives of patients diagnosed clinically was more than double that (53%), despite unstructured provision of genetic information. A substantial number of untested relatives had undiagnosed iron overload. Overall efficacy of population screening for haemochromatosis is undermined by these observations.

Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome.

MECP2 mutations are responsible for Rett syndrome (RTT). Approximately a quarter of classic RTT cases, however, do not have an identifiable mutation of the MECP2 gene. We hypothesized that larger deletions arising from a deletion prone region (DPR) occur commonly and are not being routinely detected by the current PCR-mediated screening strategies. We developed and applied a quantitative PCR strategy (qPCR) to samples referred for diagnostic assessment from 140 patients among whom RTT was strongly suspected and from a second selected group of 31 girls with classical RTT. Earlier MECP2 mutation screening in both groups of patients had yielded a wild-type result. We identified 10 large deletions (7.1%) within the first group and five deletions in the second group (16.1%). Sequencing of the breakpoints in 11 cases revealed that eight cases had one breakpoint within the DPR. Among seven cases, the breakpoint distant to the DPR involved one of several Alu repeats. Sequence analysis of the junction sequences revealed that eight cases had complex rearrangements. Examination of the MECP2 genomic sequence reveals that it is highly enriched for repeat elements, with the content of Alu repeats rising to 27.8% in intron 2, in which there was an abundance of breakpoints among our patients. Furthermore, a perfect chi sequence, known to be recombinogenic in E. coli, is located in the DPR. We propose that the chi sequence and Alu repeats are potent factors contributing to genomic rearrangement. We suggest that routine mutation screening in MECP2 should include quantitative analysis of the genomic sequences flanking the DPR.

Large deletions in the polycystic kidney disease 1 (PKD1) gene.

Since identification of the genes mutated in patients with Autosomal Dominant Polycystic Kidney Disease, PKD1 and PKD2, a large number of different germ line mutations in both genes have been found by conventional PCR-based mutation detection methods. Nevertheless, in approximately 40% of the PKD1 families the disease-causing mutation remains to be elucidated. Complex germ line rearrangements are often not detectable by these standard diagnostic techniques. To detect large deletions in the PKD1 gene we performed Field Inversion Gel Electrophoresis (FIGE) followed by Southern blot analysis with probes selected in the unique and in the reiterated region of this gene. Our analysis revealed 4 deletions in 125 patients, indicating that large deletions in PKD1 are rare. Likely, patients with a deletion that also affects the neighbouring Tuberous Sclerosis Complex 2 (TSC2) gene will be diagnosed as patients with tuberous sclerosis. It was speculated that the exceptional polypyrimidine tract located in intron 21 and the small tract in intron 22, might play a role in the pathogenesis of ADPKD. Since this region is extremely difficult to amplify by PCR, we analysed the 5.8 kb BamHI fragment that contains the polypyrimidine tracts. We did not observe a disease-linked alteration although we detected two different rare variants either in PKD1 or in one of its homologues.

Brief report: do the nature of communication impairments in autism spectrum disorders relate to the broader autism phenotype in parents?

Extensive empirical evidence indicates that the lesser variant of Autism Spectrum Disorders (ASD) involves a communication impairment that is similar to, but milder than, the deficit in clinical ASD. This research explored the relationship between the broader autism phenotype (BAP) among parents, an index of genetic liability for ASD, and proband communication difficulties. ASD probands with at least one BAP parent (identified using the Autism Spectrum Quotient) had greater structural and pragmatic language difficulties (assessed using the Children's Communication Checklist-2) than ASD probands with no BAP parent. This finding provides support for the position that genetic liability for ASD is associated with increased communication difficulties across structural and pragmatic domains.

Quality standards and samples in genetic testing.

The most critical performance indicator for medical laboratories is the delivery of accurate test results. In any laboratory, there is always the possibility that random or systematic errors may occur and place human health and welfare at risk. Laboratory quality assurance programmes continue to drive improvements in analytical accuracy. The most rigorously scrutinised data on laboratory errors, which come from transfusion medicine, reveal that the incidence of analytical errors has fallen to levels where most of the residual risk is now found in preanalytical links in the chain from patient to result, particularly activities associated with ordering of tests and sample collection. This insight is important for genetic testing because, like pretransfusion testing of patients with unknown blood groups, a substantial proportion of genotyping results cannot be immediately verified. An increasing number of clinical decisions, associated personal and social choices, and legal outcomes are now influenced by genetic test results in the absence of other confirmatory data. An incorrect test result may lead to unnecessary and irreversible interventions, which may in themselves have associated risks for the patient, inaccurate risk assessment regarding the disease, missed opportunities for disease prevention or even wrongful conviction in a court of law. Unfortunately, there is limited information available about the risk of preanalytical errors associated with, and few published guidelines regarding, sample collection for genetic testing. The growing number and range of important decisions made on the basis of genetic findings warrant a reappraisal of current standards to minimise risks in genetic testing.

SNP-based arrays complement classic cytogenetics in the detection of chromosomal aberrations in Wilms' tumor.

Wilms' tumors have characteristic chromosomal abnormalities, such as the 11p13 deletion, in a subset of cases. This is one of the very few reports comparing single nucleotide polymorphism (SNP) array analysis with conventional karyotyping of Wilms' tumors. A total of 43 frozen tumor samples were analyzed using the Affymetrix Cytogenetics Whole-Genome 2.7M array. The findings from the SNP array analysis were then compared with those from conventional karyotyping. A comparison between SNP array and conventional karyotype findings was possible in 38 of 43 specimens (88.4%). The SNP array and classic cytogenetic results were concordant in 33 of 38 specimens (87%). SNP array analysis was able to support the findings of classic cytogenetics. The SNP array detected regions of loss of heterozygosity (LOH) in 41 of 43 (95%) specimens. However, it did not detect balanced translocations and inversions that were observed by conventional cytogenetics. Our results show that the data generated from these platforms are complementary. The SNP array also detected additional gains and losses as well as regions of LOH with associated disomy, which are likely to represent segmental uniparental disomy. The observed discrepancies can be explained by the inherent limitations of each technique.