Tracy Tucker

Massively Parallel Sequencing: The Next Big Thing in Genetic Medicine

Massively parallel sequencing has reduced the cost and increased the throughput of genomic sequencing by more than three orders of magnitude, and it seems likely that costs will fall and throughput improve even more in the next few years. Clinical use of massively parallel sequencing will provide a way to identify the cause of many diseases of unknown etiology through simultaneous screening of thousands of loci for pathogenic mutations and by sequencing biological specimens for the genomic signatures of novel infectious agents. In addition to providing these entirely new diagnostic capabilities, massively parallel sequencing may also replace arrays and Sanger sequencing in clinical applications where they are currently being used. Routine clinical use of massively parallel sequencing will require higher accuracy, better ways to select genomic subsets of interest, and improvements in the functionality, speed, and ease of use of data analysis software. In addition, substantial enhancements in laboratory computer infrastructure, data storage, and data transfer capacity will be needed to handle the extremely large data sets produced. Clinicians and laboratory personnel will require training to use the sequence data effectively, and appropriate methods will need to be developed to deal with the incidental discovery of pathogenic mutations and variants of uncertain clinical significance. Massively parallel sequencing has the potential to transform the practice of medical genetics and related fields, but the vast amount of personal genomic data produced will increase the responsibility of geneticists to ensure that the information obtained is used in a medically and socially responsible manner.

Mechanisms of X-chromosome inactivation.

Mammalian X-chromosome inactivation is an impressive example of epigenetic gene regulation, whereby the majority of genes on the approximately 160 Mb X chromosome are silenced in a strictly cis-limited fashion. In this review we will discuss the important players involved in the silencing process. The process is initiated by transcription and cis-localization of the non-coding XIST RNA, which then recruits many of the epigenetic features generally associated with heterochromatin, including histone modifications, histone variants and DNA methylation.

Longitudinal study of neurofibromatosis 1 associated plexiform neurofibromas

Background: Plexiform neurofibromas are benign tumours that occur in more than half of people with neurofibromatosis 1 (NF1). These tumours can cause serious complications and can also progress to malignant peripheral nerve sheath tumours (MPNSTs), one of the leading causes of death among NF1 patients. Plexiform neurofibromas are clinically heterogeneous, and knowledge of their natural history is limited. In order to characterise the growth of plexiform neurofibromas better, we performed serial magnetic resonance imaging (MRI) in NF1 patients with such tumours. Methods: MRI was done on 44 plexiform neurofibromas in 34 NF1 patients (median age 10 years; range 1–47 years). Each tumour was measured in two dimensions from the MRI scan, and the area and growth rate were calculated. The median length of follow-up was 6 years, with an average interval of 3 years between scans. Results: 36 tumours remained stable in size throughout the period of follow-up. 8 tumours increased in size; all occurred in patients who were under 21 years of age when first studied. The single exception was a man who developed rapid tumour growth and pain in a plexiform neurofibroma that had been followed for 10 years. Biopsy showed the presence of an MPNST. Conclusion: Longitudinal MRI is a valuable means of monitoring the growth of plexiform neurofibromas in individuals with NF1.

Bone health and fracture rate in individuals with neurofibromatosis 1 (NF1)

Background: Patients with neurofibromatosis 1 (NF1) are shorter than expected and often have low bone mineral density (BMD), but the pathogenesis of these bony problems is poorly understood. Methods: We performed an exploratory study of BMD, 18 laboratory measures of bone metabolism, and fracture history in 72 adult NF1 patients. Results: Eight of the 18 clinical biochemical measures of bone health had at least 10% of NF1 patients outside the standard reference range. Serum 25-hydroxy-vitamin D concentrations were low in 56% of the NF1 patients, serum parathyroid hormone (PTH) concentrations were high in 34%, and urine deoxypyridinoline cross-link concentrations were high in 50%. Mean serum 25-hydroxy-vitamin D concentrations were significantly lower in people with NF1 than in season matched controls in both summer (p = 0.008) and winter (p<0.001). 36 (50%) of the 72 people with NF1 studied had BMD consistent with osteopenia, and 14 (19%) had BMD consistent with osteoporosis. High serum PTH concentration, high serum bone tartrate resistant acid phosphatase concentration, and high serum calcium concentration were associated with lower BMD among the NF1 patients. Males were more likely than females to have low BMD. The reported frequency of fractures in individuals with NF1 was much higher than in their unaffected siblings and spouses (p<0.001), and pathological fractures were reported only in NF1 patients. Conclusion: People with NF1 often have a generalised abnormality of bone metabolism. Further studies are needed to determine the biochemical and molecular basis of this abnormality.

Pathogenesis of hereditary tumors: beyond the “two‐hit” hypothesis

Knudsons ‘two‐hit’ hypothesis has provided extremely important insights into the pathogenesis of tumors in autosomal dominant tumor predisposition syndromes, but recent evidence suggests that some such tumors may occur without a ‘second hit’ or require more than two mutations. Inactivation of both RB1 alleles appears to be insufficient by itself to cause malignancy in the tumors that develop in patients with hereditary retinoblastoma. On the other hand, certain tumors in patients with tuberous sclerosis complex appear to develop in haploinsufficient tissues that do not have ‘second hit’ mutations of a tuberous sclerosis gene. The molecular pathogenesis of certain other tumors in patients with tuberous sclerosis complex or neurofibromatosis 1 may not be fully explained by the ‘two‐hit’ hypothesis either. Hereditary tumors, like non‐hereditary tumors, may arise by a variety of molecular mechanisms, with loss of both alleles of a particular tumor suppressor gene being a frequent, but not invariably necessary or sufficient, event. Four models are presented to explain how various tumors may arise in patients with inherited tumor predisposition syndromes such as hereditary retinoblastoma, tuberous sclerosis complex or neurofibromatosis 1. Even tumors of one particular type may develop by more than one mechanism.

Comparison of genome-wide array genomic hybridization platforms for the detection of copy number variants in idiopathic mental retardation

BackgroundClinical laboratories are adopting array genomic hybridization as a standard clinical test. A number of whole genome array genomic hybridization platforms are available, but little is known about their comparative performance in a clinical context.MethodsWe studied 30 children with idiopathic MR and both unaffected parents of each child using Affymetrix 500 K GeneChip SNP arrays, Agilent Human Genome 244 K oligonucleotide arrays and NimbleGen 385 K Whole-Genome oligonucleotide arrays. We also determined whether CNVs called on these platforms were detected by Illumina Hap550 beadchips or SMRT 32 K BAC whole genome tiling arrays and tested 15 of the 30 trios on Affymetrix 6.0 SNP arrays.ResultsThe Affymetrix 500 K, Agilent and NimbleGen platforms identified 3061 autosomal and 117 X chromosomal CNVs in the 30 trios. 147 of these CNVs appeared to be de novo, but only 34 (22%) were found on more than one platform. Performing genotype-phenotype correlations, we identified 7 most likely pathogenic and 2 possibly pathogenic CNVs for MR. All 9 of these putatively pathogenic CNVs were detected by the Affymetrix 500 K, Agilent, NimbleGen and the Illumina arrays, and 5 were found by the SMRT BAC array. Both putatively pathogenic CNVs identified in the 15 trios tested with the Affymetrix 6.0 were identified by this platform.ConclusionsOur findings demonstrate that different results are obtained with different platforms and illustrate the trade-off that exists between sensitivity and specificity. The large number of apparently false positive CNV calls on each of the platforms supports the need for validating clinically important findings with a different technology.

Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes

Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis.

Uniparental disomy: can SNP array data be used for diagnosis?

Purpose:Single-nucleotide polymorphism microarray analysis identifies copy-number variants and blocks of homozygosity, suggestive of consanguinity or uniparental disomy. The purpose of this study was to validate chromosomal microarray analysis for the identification of uniparental disomy in a clinical laboratory.Methods:In phase I of this retrospective study, nine cases with uniparental disomy for chromosomes 7 (n = 1), 14 (n = 1), and 15 (n = 7), identified by conventional polymorphic microsatellite marker analysis were analyzed on the Affymetrix 6.0 single-nucleotide polymorphism array. In phase II, four cases of uniparental disomy 15 showing heterozygosity for all microsatellite markers were analyzed using the same array.Results:Chromosomal microarray analysis detected blocks of homozygosity in eight of the nine cases in phase I. Phase II analysis of molecularly defined heterodisomy failed to detect blocks of homozygosity in three of the four cases. The four cases in which microarray did not detect blocks of homozygosity all involved chromosome 15.Conclusion:A failure to recombine may predispose to nondisjunction and, therefore, to uniparental disomy. Four cases of heterodisomy 15 were not detected by array, suggesting a lack of recombination. Therefore, a normal chromosomal microarray result for chromosome 15 does not exclude the possibility of uniparental disomy. This observation may apply to other chromosomes; however, further study is needed.Genet Med 2012:14(8):753–756

A co-occurrence of osteogenesis imperfecta type VI and cystinosis.

Osteogenesis imperfecta type VI (OI type VI) is a rare autosomal recessive disorder caused by mutations in the SERPINF1 gene that encodes pigment epithelium‐derived factor (PEDF). Cystinosis is an autosomal recessive lysosomal transport disorder caused by mutations in the CTNS gene. Both SERPINF1 and CTNS are located on chromosome 17p13.3. We describe an individual presenting with both OI type VI and cystinosis. The patient was diagnosed with cystinosis at the age of 11 months and OI type VI on bone biopsy at the age of 8 years. He has sustained over 30 fractures during his lifetime, and at the age of 19 years entered end‐stage renal disease and subsequent renal transplant. An Affymetrix 6.0 array was used to look for areas of loss of heterozygosity on chromosome 17. Sequencing of the SERPINF1 and CTNS genes was performed, followed by quantitative PCR and Western blot of PEDF to characterize the identified mutation. A 6.58 Mb region of homozygosity was identified on the Affymetrix 6.0 array, encompassing both the SERPINF1 and CTNS genes. Sequencing of the genes identified homozygosity for a known pathogenic CTNS mutation and for a novel in‐frame duplication in SERPINF1. Skin fibroblasts produced a markedly reduced amount of SERPINF1 transcript and PEDF protein. This patient has the concurrent phenotype of two rare recessive diseases, cystinosis and OI type VI. We identified for the first time an in‐frame duplication in SERPINF1 that is responsible for the OI type VI phenotype in this patient.

Increased dental caries in people with neurofibromatosis 1

Neurofibromatosis 1 (NF1) is an autosomal dominant disease that is associated with multiple café‐au‐lait spots and neurofibromas. Low bone mineral density is frequent in people with NF1, and focal boney abnormalities are characteristic but uncommon features. Dental abnormalities can occur in association with oral neurofibromas but have not otherwise been described in people with NF1. Questionnaires regarding dental caries were sent to families that included at least one individual with NF1. Siblings with NF1 reported significantly more dental caries (mean ± SD, 8.1 ± 6.6) than siblings without NF1 in these families (5.5 ± 5.8, p = 0.019). Our findings suggest that dental caries occur more frequently than expected among people with NF1 and that individuals with this condition may require a modified dental care program.