Determining Cost-Optimal Next-Generation Sequencing Panels for Rare Disease and Pharmacogenomics Testing.

BACKGROUND: Multi-gene panel sequencing using next-generation sequencing (NGS) methods is a key tool for genomic medicine. However, with an estimated 140 000 genomic tests available, current system inefficiencies result in high genetic-testing costs. Reduced testing costs are needed to expand the availability of genomic medicine. One solution to improve efficiency and lower costs is to calculate the most cost-effective set of panels for a typical pattern of test requests. METHODS: We compiled rare diseases, associated genes, point prevalence, and test-order frequencies from a representative laboratory. We then modeled the costs of the relevant steps in the NGS process in detail. Using a simulated annealing-based optimization procedure, we determined panel sets that were more cost-optimal than whole exome sequencing (WES) or clinical exome sequencing (CES). Finally, we repeated this methodology to cost-optimize pharmacogenomics (PGx) testing. RESULTS: For rare disease testing, we show that an optimal choice of 4-6 panels, uniquely covering genes that comprise 95% of the total prevalence of monogenic diseases, saves $257-304 per sample compared with WES, and $66-135 per sample compared with CES. For PGx, we show that the optimal multipanel solution saves $6-7 (27%-40%) over a single panel covering all relevant gene-drug associations. CONCLUSIONS: Laboratories can reduce costs using the proposed method to obtain and run a cost-optimal set of panels for specific test requests. In addition, payers can use this method to inform reimbursement policy.

Spectrin mutations cause spinocerebellar ataxia type 5.

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

A review of genetic counseling for Charcot Marie Tooth disease (CMT).

Charcot Marie Tooth disease (CMT) encompasses the inherited peripheral neuropathies. While four genes have been found to cause over 90 % of genetically identifiable causes of CMT (PMP22, GJB1, MPZ, MFN2), at least 51 genes and loci have been found to cause CMT when mutated, creating difficulties for clinicians to find a genetic subtype for families. Here, the classic features of CMT as well as characteristic features of the most common subtypes of CMT are described, as well as methods for narrowing down the possible subtypes. Psychosocial concerns particular to the CMT population are identified. This is the most inclusive publication for CMT-specific genetic counseling.

Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort.

Mutations in the DMD gene, encoding the dystrophin protein, are responsible for the dystrophinopathies Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and X-linked Dilated Cardiomyopathy (XLDC). Mutation analysis has traditionally been challenging, due to the large gene size (79 exons over 2.2 Mb of genomic DNA). We report a very large aggregate data set comprised of DMD mutations detected in samples from patients enrolled in the United Dystrophinopathy Project, a multicenter research consortium, and in referral samples submitted for mutation analysis with a diagnosis of dystrophinopathy. We report 1,111 mutations in the DMD gene, including 891 mutations with associated phenotypes. These results encompass 506 point mutations (including 294 nonsense mutations) and significantly expand the number of mutations associated with the dystrophinopathies, highlighting the utility of modern diagnostic techniques. Our data supports the uniform hypermutability of CGA>TGA mutations, establishes the frequency of polymorphic muscle (Dp427m) protein isoforms and reveals unique genomic haplotypes associated with "private" mutations. We note that 60% of these patients would be predicted to benefit from skipping of a single DMD exon using antisense oligonucleotide therapy, and 62% would be predicted to benefit from an inclusive multiexonskipping approach directed toward exons 45 through 55.

A focal domain of extreme demethylation within D4Z4 in FSHD2.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disease with an unclear genetic mechanism. Most patients have a contraction of the D4Z4 macrosatellite repeat array at 4qter, which is thought to cause partial demethylation (FSHD1) of the contracted allele. Demethylation has been surveyed at 3 restriction enzyme sites in the first repeat and only a single site across the entire array, and current models postulate that a generalized D4Z4 chromatin alteration causes FSHD. The background of normal alleles has confounded the study of epigenetic alterations; however, rare patients (FSHD2) have a form of the disease in which demethylation is global, i.e., on all D4Z4 elements throughout the genome. Our objective was to take advantage of the global nature of FSHD2 to identify where disease-relevant methylation changes occur within D4Z4. METHODS: Using bisulfite sequencing of DNA from blood and myoblast cells, methylation levels at 74 CpG sites across 3 disparate regions within D4Z4 were measured in FSHD2 patients and controls. RESULTS: We found that rates of demethylation caused by FSHD2 are not consistent across D4Z4. We identified a focal region of extreme demethylation within a 5' domain, which we named DR1. Other D4Z4 regions, including the DUX4 ORF, were hypomethylated but to a much lesser extent. CONCLUSIONS: These data challenge the simple view that FSHD is caused by a broad "opening" of D4Z4 and lead us to postulate that the region of focal demethylation is the site of action of the key D4Z4 chromatin regulatory factors that go awry in FSHD.

Motor and cognitive assessment of infants and young boys with Duchenne Muscular Dystrophy: results from the Muscular Dystrophy Association DMD Clinical Research Network.

Therapeutic trials in Duchenne Muscular Dystrophy (DMD) exclude young boys because traditional outcome measures rely on cooperation. The Bayley III Scales of Infant and Toddler Development (Bayley III) have been validated in developing children and those with developmental disorders but have not been studied in DMD. Expanded Hammersmith Functional Motor Scale (HFMSE) and North Star Ambulatory Assessment (NSAA) may also be useful in this young DMD population. Clinical evaluators from the MDA-DMD Clinical Research Network were trained in these assessment tools. Infants and boys with DMD (n = 24; 1.9 ± 0.7 years) were assessed. The mean Bayley III motor composite score was low (82.8 ± 8; p ≤ .0001) (normal = 100 ± 15). Mean gross motor and fine motor function scaled scores were low (both p ≤ .0001). The mean cognitive comprehensive (p=.0002), receptive language (p ≤ .0001), and expressive language (p = .0001) were also low compared to normal children. Age was negatively associated with Bayley III gross motor (r = -0.44; p = .02) but not with fine motor, cognitive, or language scores. HFMSE (n=23) showed a mean score of 31 ± 13. NSAA (n = 18 boys; 2.2 ± 0.4 years) showed a mean score of 12 ± 5. Outcome assessments of young boys with DMD are feasible and in this multicenter study were best demonstrated using the Bayley III.

Cerebral and muscle MRI abnormalities in myotonic dystrophy.

Pathophysiological mechanisms underlying the clinically devastating CNS features of myotonic dystrophy (DM) remain more enigmatic and controversial than do the muscle abnormalities of this common form of muscular dystrophy. To better define CNS and cranial muscle changes in DM, we used quantitative volumetric and diffusion tensor MRI methods to measure cerebral and masticatory muscle differences between controls (n=5) and adults with either congenital (n=5) or adult onset (n=5) myotonic dystrophy type 1 and myotonic dystrophy type 2 (n=5). Muscle volumes were diminished in DM1 and strongly correlated with reduced white matter integrity and gray matter volume. Moreover, correlation of reduced fractional anisotropy (white matter integrity) and gray matter volume in both DM1 and DM2 suggests that these abnormalities may share a common underlying pathophysiological mechanism. Further quantitative temporal and spatial characterization of these features will help delineate developmental and progressive neurological components of DM, and help determine the causative molecular and cellular mechanisms.

SNP haplotype mapping in a small ALS family.

The identification of genes for monogenic disorders has proven to be highly effective for understanding disease mechanisms, pathways and gene function in humans. Nevertheless, while thousands of Mendelian disorders have not yet been mapped there has been a trend away from studying single-gene disorders. In part, this is due to the fact that many of the remaining single-gene families are not large enough to map the disease locus to a single site in the genome. New tools and approaches are needed to allow researchers to effectively tap into this genetic gold-mine. Towards this goal, we have used haploid cell lines to experimentally validate the use of high-density single nucleotide polymorphism (SNP) arrays to define genome-wide haplotypes and candidate regions, using a small amyotrophic lateral sclerosis (ALS) family as a prototype. Specifically, we used haploid-cell lines to determine if high-density SNP arrays accurately predict haplotypes across entire chromosomes and show that haplotype information significantly enhances the genetic information in small families. Panels of haploid-cell lines were generated and a 5 centimorgan (cM) short tandem repeat polymorphism (STRP) genome scan was performed. Experimentally derived haplotypes for entire chromosomes were used to directly identify regions of the genome identical-by-descent in 5 affected individuals. Comparisons between experimentally determined and in silico haplotypes predicted from SNP arrays demonstrate that SNP analysis of diploid DNA accurately predicted chromosomal haplotypes. These methods precisely identified 12 candidate intervals, which are shared by all 5 affected individuals. Our study illustrates how genetic information can be maximized using readily available tools as a first step in mapping single-gene disorders in small families.

Myotonic dystrophy type 2: human founder haplotype and evolutionary conservation of the repeat tract.

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning >700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.

Spinocerebellar ataxia type 8: molecular genetic comparisons and haplotype analysis of 37 families with ataxia.

We reported elsewhere that an untranslated CTG expansion causes the dominantly inherited neurodegenerative disorder spinocerebellar ataxia type 8 (SCA8). SCA8 shows a complex inheritance pattern with extremes of incomplete penetrance, in which often only one or two affected individuals are found in a given family. SCA8 expansions have also been found in control chromosomes, indicating that separate genetic or environmental factors increase disease penetrance among SCA8-expansion-carrying patients with ataxia. We describe the molecular genetic features and disease penetrance of 37 different families with SCA8 ataxia from the United States, Canada, Japan, and Mexico. Haplotype analysis using 17 STR markers spanning an approximately 1-Mb region was performed on the families with ataxia, on a group of expansion carriers in the general population, and on psychiatric patients, to clarify the genetic basis of the reduced penetrance and to investigate whether CTG expansions among different populations share a common ancestral background. Two major ancestrally related haplotypes (A and A') were found among white families with ataxia, normal controls, and patients with major psychosis, indicating a common ancestral origin of both pathogenic and nonpathogenic SCA8 expansions among whites. Two additional and distinct haplotypes were found among a group of Japanese families with ataxia (haplotype B) and a Mexican family with ataxia (haplotype C). Our finding that SCA8 expansions on three independently arising haplotypes are found among patients with ataxia and cosegregate with ataxia when multiple family members are affected further supports the direct role of the CTG expansion in disease pathogenesis.