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Massively parallel sequencing has tremendous diagnostic potential but requires enriched templates for sequencing. Here we report the validation of an arraybased sequence capture method in genetically heterogeneous disorders. The model disorder selected was AR ataxia, using five subjects with known mutations and two unaffected controls. The genomic sequences of seven disease genes, together with two control loci were targeted on a 2-Mb sequence-capture array. After enrichment, the patients' DNA samples were analyzed using one-quarter Roche GS FLX Titanium sequencing run, resulting in an average of 65Mb of sequence data per patient. This was sufficient for an average 25-fold coverage/base in all targeted regions. Enrichment showed high specificity; on average, 80% of uniquely mapped reads were on target. Importantly, this approach enabled automated detection of deletions and hetero- and homozygous point mutations for 6/7 mutant alleles, and greater than 99% accuracy for known SNP variants. Our results also clearly show reduced coverage for sequences in repeat-rich regions, which significantly impacts the reliable detection of genomic variants. Based on these findings we recommend a minimal coverage of 15-fold for diagnostic implementation of this technology. We conclude that massive parallel sequencing of enriched samples enables personalized diagnosis of heterogeneous genetic disorders and qualifies for rapid diagnostic implementation. Refereed/Peer-reviewed

Contains fulltext : 88828.pdf (Publisher’s version ) (Open Access) BACKGROUND: A significant component of the variation in cognitive disability that is observed in Duchenne muscular dystrophy (DMD) is known to be under genetic regulation. In this study we report correlations between standardised measures of intelligence and mutational class, mutation size, mutation location and the involvement of dystrophin isoforms. METHODS AND RESULTS: Sixty two male subjects were recruited as part of a study of the cognitive spectrum in boys with DMD conducted at the Sydney Children's Hospital (SCH). All 62 children received neuropsychological testing from a single clinical psychologist and had a defined dystrophin gene (DMD) mutation; including DMD gene deletions, duplications and DNA point mutations. Full Scale Intelligence Quotients (FSIQ) in unrelated subjects with the same mutation were found to be highly correlated (r = 0.83, p = 0.0008), in contrast to results in previous publications. In 58 cases (94%) it was possible to definitively assign a mutation as affecting one or more dystrophin isoforms. A strong association between the risk of cognitive disability and the involvement of groups of DMD isoforms was found. In particular, improvements in the correlation of FSIQ with mutation location were identified when a new classification system for mutations affecting the Dp140 isoform was implemented. SIGNIFICANCE: These data represent one of the largest studies of FSIQ and mutational data in DMD patients and is among the first to report on a DMD cohort which has had both comprehensive mutational analysis and FSIQ testing through a single referral centre. The correlation between FSIQ results with the location of the dystrophin gene mutation suggests that the risk of cognitive deficit is a result of the cumulative loss of central nervous system (CNS) expressed dystrophin isoforms, and that correct classification of isoform involvement results in improved estimates of risk.

Contains fulltext : 81613.pdf (Publisher’s version ) (Closed access) Pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID) syndrome is a recently described autosomal recessive disorder associated with predominantly antibody negative, insulin-dependent diabetes mellitus. In order to identify the genetic basis of PHID and study its relationship with glucose metabolism, we performed homozygosity mapping in five unrelated families followed by candidate gene sequencing. Five loss-of-function mutations were identified in the SLC29A3 gene which encodes a member of a highly conserved protein family that transports nucleosides, nucleobases and nucleoside analogue drugs, hENT3. We show that PHID is allelic with a related syndrome without diabetes mellitus, H syndrome. The interaction of SLC29A3 with insulin signaling pathways was then studied using an established model in Drosophila melanogaster. Ubiquitous knockdown of the Drosophila ortholog of hENT3, dENT1 is lethal under stringent conditions; whereas milder knockdown induced scutellar bristle phenotypes similar to those previously reported in the knockdown of the Drosophila ortholog of the Islet gene. A cellular growth assay showed a reduction of cell size/number which could be rescued or enhanced by manipulation of the Drosophila insulin receptor and its downstream signaling effectors, dPI3K and dAkt. In summary, inactivating mutations in SLC29A3 cause a syndromic form of insulin-dependent diabetes in humans and in Drosophila profoundly affect cell size/number through interactions with the insulin signaling pathway. These data suggest that further investigation of the role of SLC29A3 in glucose metabolism is a priority for diabetes research.

Summary We describe the clinical experience of a male patient with acute promyelocytic leukaemia, relapsing after sex-mismatched allogeneic bone marrow transplantation and treated with all-trans-retinoic acid. Resolution of the coagulopathy was observed by day 7 of therapy. A complete remission was achieved by day 47 after a period of pancytopenia, dysplastic myeloid maturation and bone marrow hypo-cellularity with necrosis and fibrosis. Serial cytogenetic analyses revealed a progressive loss of the male leukaemic clone [46XY, t(15;17)] and emergence of normal female (donor) cells [46XX] which became completely dominant with remission. Adverse effects of all-trans-retinoic acid included bone pain and a prominent leucocytosis requiring leukaphereses and hydroxyurea therapy. All-trans-retinoic acid can induce complete remission of recurrent acute promyelocytic leukaemia following bone marrow transplantation. The data suggest that remission is due to differentiation and suppression of the leukaemic clone while allowing repopulation of the marrow with non-malignant cells.

Contains fulltext : 71488.pdf (Publisher’s version ) (Closed access)

The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia.

Objectives We present the first prenatal diagnosis of familial hepatic veno-occlusive disease with immunodeficiency (VODI). Homozygous mutations in the gene SP110 are the genetic basis of VODI. The proband in this report presented at three months of age with hepatomegaly hepatic failure and was found to have hypogammaglobulinemia. He died one month after hepatic transplant at eight months of age due to hemophagocytic syndrome. DNA testing detected a homozygous truncating mutation in exon 5; SP110 c.642delC. Prenatal testing was offered to this family in a subsequent pregnancy. Methods Chorion villus was sampled at 12 weeks' gestation. DNA was extracted using standard techniques, and sequencing of SP110 exon 5 was performed using flanking primers. Maternal contamination was excluded by examining STR markers in CVS and maternal DNA. Results A heterozygous SP110 c.642delC mutation was detected in exon 5. This mutation was present in heterozygous form in both parents. Conclusions The prenatal test result is predictive of a child with a normal immune and hepatic phenotype. This report presents the first prenatal molecular diagnosis for VODI and shows the importance of molecular genetic research in not only defining the aetiology of syndromes but also in assisting reproductive choices through the collaboration of genetic and feto-maternal services. Copyright © 2007 John Wiley & Sons, Ltd.

Comparison between BRCA1-associated and sporadic ovarian carcinomas is a potential method to identify candidate modifier gene/s involved in the carcinogenic pathway of either or both groups. A previous study identified a significant difference in the frequency of copy number gain at 2q24-q32 by comparing BRCA1-associated and sporadic ovarian tumour specimens using comparative genomic hybridisation (CGH). The present study aimed to investigate the reported allelic imbalance at 2q24-32 by amplification of several microsatellite markers at the region by quantitative microsatellite analysis (QuMA) using Taqman at the same region identified as a site of allelic imbalance.The copy number of the genomic region in 2q24-32 was established in 21 BRCA1-associated ovarian carcinomas and 14 sporadic cases using quantitative microsatellite polymerase chain reaction (PCR). Statistical analysis was performed using permutation test analysis.A significant loss at D2S156 marker (2q24.2) (p = 0.026) compared with the other three markers at 2q24-32 was found in the sporadic cohort but not in the BRCA1-associated group (p = 0.385).Our data do not support the association between copy number gain at 2q24-32 and BRCA1 mutation status in ovarian cancers reported previously. The novel finding of the present study was significant loss at 2q24.2 in sporadic ovarian cancers.

We present a case of Beare-Stevenson syndrome with a broad range of phenotypic features including craniosynostosis, cutis gyrata, choanal stenosis, bifid scrotum with perineal hypospadias and a caudal appendage. The paternal age at the time of conception was 62 years consistent with a paternal age effect. Mutation analysis was undertaken and demonstrated the FGFR2 Y375C mutation. This case, one of only nine with molecular analysis, confirms the significant morbidity associated with this syndrome.

Atrial septal defect (ASD) is a common congenital heart disease (CHD) occurring in 5 to 7 per 10 000 live births. Mutations in 5 human genes ( NKX2.5 , TBX5 , GATA4 , MYHC , ACTC ) are known to cause dominant ASD, but these account for a minority of cases. Human and mouse data suggest that ASD exists in an anatomical continuum with milder septal variants patent foramen ovale (PFO) and atrial septal aneurysm, strongly associated with ischemic stroke and migraine. We have previously shown in inbred mice that the incidence of PFO strongly correlates with length of the interatrial septum primum , defining a quantitative trait underlying PFO risk. To better understand genetic causation of atrial septal abnormalities, we mapped quantitative trait loci (QTL) influencing septal morphology using mouse strains (QSi5 and 129T2/SvEms) maximally informative for PFO incidence and 3 quantitative septal anatomical traits including septum primum length. [QSi5×129T2/SvEms]F2 intercross animals (n=1437) were phenotyped and a whole genome scan performed at an average 17-cM interval. Statistical methodology scoring PFO as a binary phenotype was developed as a confirmatory mapping technique. We mapped 7 significant and 6 suggestive QTL modifying quantitative phenotypes, with 4 supported by binary analysis. Quantitative traits, although strongly associated with PFO ( P

In this communication we describe the clinical and molecular genetic findings in a family with a variable ectrodactyly linked to SHFM3. This is only the second detailed report of the clinical features of the SHFM3 linked syndrome in a large pedigree. Within this family the expressivity of the condition ranges from the classical ectrodactyly deformity to partial absence of the thumb and agenesis of the distal tip of the index finger. There is discordant limb severity, with the feet more severely affected than the hands. Two individuals have a nail dysplasia indicating the presence of a minor ectodermal component. A cleft palate was present in one individual. Radiological features of family members include short metacarpals with rounded proximal heads, agenesis of the radial ray, epiphysial coning, and an unusual supernumerary ossicle opposed to the distal phalanx of the left thumb. Genetic mapping studies in this family exclude p63 involvement and demonstrate that ectrodactyly in this pedigree is linked to the SHFM3 region on chromosome 10q24. A meiotic recombination event enabled exclusion of a maximum of 1.9 Mb of DNA from the previously known critical region thereby narrowing the critical interval to between D10S1265 and D10S222, with the minimal critical region being between D10S1240 and D10S1267. Further investigations are in progress to identify the gene within the SHFM3 critical region responsible for ectrodactyly.

The sequential transcriptional activation of cyclins, the regulatory subunits of cell-cycle-specific kinases, is thought to regulate progress through the cell cycle. Cyclins are therefore potential oncogenes, and cyclin D1 overexpression and/or amplification at its genomic locus, 11q13, are common features of several human cancers. Induction of cyclin D1 is an early response to mitogenic stimulation in several cell types, but the consequences of altered expression of this gene in human cells of epithelial origin remain undefined. We assessed the effects of alterations of cyclin D1 expression in human breast cancer cells by generating T-47D cells expressing human cyclin D1 under the control of a zinc-responsive metallothionein promoter. In cycling cells induction of cyclin D1 after zinc treatment resulted in an increase in the number of cells progressing through G1 and in the rate of transition from G1 to S phase, indicating that cyclin D1 is rate-limiting for progress through G1 phase. In cells arrested in early G1 phase after growth factor deprivation, zinc induction of cyclin D1 was sufficient for completion of the cell cycle, a process requiring growth factor stimulation in control cells. These data demonstrate a critical role for cyclin D1 in human breast cancer cell-cycle control and suggest that deregulated expression of cyclin D1 is likely to reduce dependence on normal physiological growth stimuli, thereby providing a growth advantage to tumor cells and a potential mechanism of resistance to endocrine therapy.

Sanger sequencing using capillary electrophoresis has been the backbone of the molecular genetics diagnostic laboratory for the better part of 20 years. Its benefits are relatively long read length (up to 1 kbp), a high standard of accuracy, and the ability to co-analyse from 1–384 samples/instrument depending on the capillary arrays employed. Capillary electrophoresis coupled with liquid handling stations permits an impressive sample through-put. The problems with Sanger/capillary sequencing are the reliance on purified clonal DNA samples as starting material, sequence costs in the range of approximately 1 cent per base with a PHRED score of 40 in the diagnostic laboratory setting, and a limited ability to accurately quantify allele frequencies. These later problems can be addressed by next generation sequencing which massively improves sequencing output by using randomly sheared DNA with oil/bead amplification and parallel sequencing. Currently MPS sequencing results require validating with Sanger sequencing.

The implementation of Next Generation Sequencing in the diagnostic laboratory presents unique opportunities and problems. For the first time it appears likely that a single instrument will be able to detect and characterise in parallel the common forms of genetic mutation and do so in a genome-wide fashion. For a diagnostic laboratory the instrumentation costs are high and the costs of consumables per run are expensive; therefore, the applications of the instrument need to be carefully designed to maximise yields of meaningful data. Issues of significance for the laboratory include data handling, accreditation and certification of bioinformaticians, standardisation of the data production pipeline and the approach to reporting the large numbers of variants identified.

Two quantitative trait loci (QTL) for mouse bodyweight have previously been reported on mouse chromosomes (MMU) 4 and 11 from crosses of a highly fecund and large mouse strain, Inbred Quackenbush-Swiss 5 (QSi5) and C57Bl/6J. QSi5 has now been crossed with CBA/CaH to produce 1128 F2 mice to confirm the existence and effect of these QTL. In total, 226 mice from the upper and lower deciles for bodyweight were genotyped using 12 microsatellite markers covering MMU4 and MMU11. Regression and maximum likelihood based interval mapping by either including all mice (ungenotyped mice were treated as having missing genotypes) or including only selectively genotyped mice in the analyses were used to estimate the positions and effects of the QTL. The results confirmed the existence and effects of both QTL. Although all methods estimated the same QTL positions, the QTL effects were overestimated compared with the estimates using a suggested method (maximum likelihood by including all mice in the analysis). However, the overestimated QTL effects could be mathematically corrected. Since the confidence intervals of both QTL are still too large for positional cloning, an advanced intercross line is being bred for finely mapping these bodyweight QTL.

A procedure for the preparation of DNA from bacteriophage λ is described, using the Beckman TL-100 bench-top ultracentrifuge. The procedure involves growth of phage in agar plates, precipitation with polyethylene glycol, and a single centrifugation in cesium chloride under conditions that disrupt the phage coat. The method avoids the use of enzymes, ion exchange resins, and phenol. It can be completed in less than a day. The resulting DNA is good purity and is easily cuttable by restriction enzymes.