James R. Lupski

Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR

ABSTRACT Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of Neisseria meningitidis infections. N. meningitidis outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.

Emergence of a predominant clone of community-acquired Staphylococcus aureus among children in Houston, Texas

Background: Community-acquired (CA), methicillin-resistant Staphylococcus aureus (MRSA) infections among children are increasing in the United States. At Texas Childrens Hospital (TCH), surveillance has been in place since August 2001. The objectives of this study were to describe the distribution of CA S. aureus among patients at TCH and to study genomic relationships of isolates collected between August 2001 and July 2003. Methods: Genomic relationships were determined with repetitive element-polymerase chain reaction (PCR). Multilocus sequence typing was performed for selected strains representing major clones. Molecular characterization of CA-MRSA was performed with PCR, including staphylococcal cassette chromosome (SCCmec), pvl (lukS-PV plus lukF-PV), hla, hlb and selected microbial surface components recognizing adhesive matrix molecule genes, ie, cna, clfA, fnbA and fnbB. Results: A 62% increase was observed in CA S. aureus infections from year 1 (2001–2002) to year 2 (2002–2003), whereas the annual number of hospital admissions was unchanged. CA methicillin-sensitive S. aureus isolates were more likely to be associated with invasive infections than were CA-MRSA isolates (P < 0.01). TCH clone A, sequence type (ST) 8, was responsible for approximately 94% of all CA-MRSA isolated from children in the greater Houston area. Clone A differed from clones B (ST30) and C (ST1) by lacking the cna gene while carrying the fnbB gene. Conclusions: One CA-MRSA clone, TCH clone A, has become the predominant cause of CA S. aureus infections among children in the Houston area. It causes a wide spectrum of diseases, including complicated pneumonia.

Newfoundland rod-cone dystrophy, an early-onset retinal dystrophy, is caused by splice-junction mutations in RLBP1.

Some isolated populations exhibit an increased prevalence of rare recessive diseases. The island of Newfoundland is a characteristic geographic isolate, settled by a small number of families primarily during the late 1700s and early 1800s. During our studies of this population, we identified a group of families exhibiting a retinal dystrophy reminiscent of retinitis punctata albescens but with a substantially lower age at onset and more-rapid and distinctive progression, a disorder that we termed Newfoundland rod-cone dystrophy (NFRCD). The size of one of these families was sufficient to allow us to perform a genomewide screen to map the NFRCD locus. We detected significant linkage to markers on the long arm of chromosome 15, in a region encompassing RLBP1, the gene encoding the cellular retinaldehyde-binding protein. Previously, mutations in RLBP1 have been associated with other retinal dystrophies, leading us to hypothesize that RLBP1 mutations might also cause NFRCD. To test this hypothesis, we sequenced all coding exons and splice junctions of RLBP1. We detected two sequence alterations, each of which is likely to be pathogenic, since each segregates with the disease and is predicted to interfere with mRNA splicing. In contrast to some previously reported RLBP1 mutations, which yield a protein that may retain some residual activity, each NFRCD mutation is likely to give rise to a null allele. This difference may account for the severe phenotype in these families and exemplifies the molecular continuum that underlies clinically distinct but genetically related entities.

Repetitive Sequence-based PCR (rep-PCR) DNA Fingerprinting of Bacterial Genomes

Bacterial chromosomes contain multiple interspersed repetitive sequences that occupy intergenic regions at sites dispersed throughout the genome. Such blocks of noncoding, repetitive sequences can serve as multiple genetic targets for oligonucleotide probes, enabling the generation of unique DNA profiles or fingerprints for individual bacterial strains. DNA fingerprinting requires the resolution of differently sized DNA fragments derived from chromosomal or plasmid DNA by restriction endonuclease-mediated digestion and/or DNA amplification to yield a band pattern that serves as a unique identifier. These unique “bar codes” or DNA fingerprints define each bacterial chromosome without the need for measuring gene expression or enzyme function. Genotypic or molecular approaches differ with respect to the level of resolution of individual bacterial species or strains into distinct categories (Figure 34-1).

Fundus albipunctatus and retinitis punctata albescens in a pedigree with an R150Q mutation in RLBP1

Fundus albipunctatus (FA; OMIM 136880) is a rare form of apparently stationary night blindness characterized by the presence of myriad symmetrical round white dots in the fundus with a greater concentration in the midperiphery. A distantly similar but distinct clinical entity, retinitis punctata albescens (RPA), is also characterized by aggregation of irregular white flecks but is progressive and evolves to generalized atrophy of the retina. We studied 4 consanguineous kindreds diagnosed with FA from Saudi Arabia. Given the substantial phenotypic variation and overlap between different flecked retinal dystrophies, we evaluated all known genes associated with such conditions by both genetic analysis and direct sequencing. In one kindred, KKESH‐099, we identified a homozygous R150Q alteration in RLBP1, the gene encoding the cellular retinaldehyde binding protein, associated previously with both recessive retinitis pigmentosa (arRP) and RPA. Examination of several patients aged 3–20 years over a 9‐year period presented no evidence for either RP or RPA. In contrast, clinical examination of individuals with the same mutation in their fourth and fifth decade revealed signs consistent with RPA. The data suggest that the R150Q mutation in RLBP1 may result in RPA with slow progression. More importantly, younger individuals diagnosed with the milder disorder FA thought to be stationary may evolve to a more devastating and progressive phenotype.

Somatic mosaicism underlies X-linked acrogigantism syndrome in sporadic male subjects

Somatic mosaicism has been implicated as a causative mechanism in a number of genetic and genomic disorders. X-linked acrogigantism (XLAG) syndrome is a recently characterized genomic form of pediatric gigantism due to aggressive pituitary tumors that is caused by submicroscopic chromosome Xq26.3 duplications that include GPR101 We studied XLAG syndrome patients (n= 18) to determine if somatic mosaicism contributed to the genomic pathophysiology. Eighteen subjects with XLAG syndrome caused by Xq26.3 duplications were identified using high-definition array comparative genomic hybridization (HD-aCGH). We noted that males with XLAG had a decreased log2ratio (LR) compared with expected values, suggesting potential mosaicism, whereas females showed no such decrease. Compared with familial male XLAG cases, sporadic males had more marked evidence for mosaicism, with levels of Xq26.3 duplication between 16.1 and 53.8%. These characteristics were replicated using a novel, personalized breakpoint junction-specific quantification droplet digital polymerase chain reaction (ddPCR) technique. Using a separate ddPCR technique, we studied the feasibility of identifying XLAG syndrome cases in a distinct patient population of 64 unrelated subjects with acromegaly/gigantism, and identified one female gigantism patient who had had increased copy number variation (CNV) threshold for GPR101 that was subsequently diagnosed as having XLAG syndrome on HD-aCGH. Employing a combination of HD-aCGH and novel ddPCR approaches, we have demonstrated, for the first time, that XLAG syndrome can be caused by variable degrees of somatic mosaicism for duplications at chromosome Xq26.3. Somatic mosaicism was shown to occur in sporadic males but not in females with XLAG syndrome, although the clinical characteristics of the disease were similarly severe in both sexes.

From genomic medicine to precision medicine: highlights of 2015

2015 has been an exciting year for genomic medicine. We asked our Section Editors to discuss the breakthroughs in their fields of expertise, and what these might mean for the future. As in previous years, exome and whole-genome sequencing are leading the way in our understanding of disease mechanisms, their diagnosis and treatment, while information about the protein products of the genome has also grown, driven by novel technological advances. Precision medicine is now taking off as an important topic in the public health sphere and in education, and no discussion of 2015 would be complete without a mention of the huge advances in gene editing technologies, the implications of which have dominated ethics and policy debate.

Interspersed Repetitive Sequences in Bacterial Genomes

Prokaryotic and eukaryotic genomes contain dispersed repetitive sequences separating longer single-copy DNA sequences (Lupski and Weinstock, 1992). Various classes of repeated DNA sequences are present in diverse prokaryotic genomes. Coding sequences, such as ribosomal RNA genes (see Chapter 21) and insertion sequences (see Chapters 4 and 20), may be repeated multiple times per genome but are usually present in relatively low copy numbers. Conserved motifs or subsequences within related genes, such as those encoding transfer RNA (tRNA), may also be repeated multiple times within individual genomes.

Xq26.3 Duplication in a Boy With Motor Delay and Low Muscle Tone Refines the X-Linked Acrogigantism Genetic Locus

We describe a 4-year-old boy with developmental delay who was found to carry by clinical grade (CG) molecular cytogenetics (MCs) a chromosome Xq26 microduplication. The report prompted a referral of the patient for possible X-linked acrogigantism (X-LAG), a well-defined condition (MIM300942) due to chromosomal microduplication of a nearby region. The patient was evaluated clinically and investigated for endocrine abnormalities related to X-LAG and not only did he not have acrogigantism, but his growth parameters and other hormones were all normal. We then performed high definition MCs and the duplication copy number variant (CNV) was confirmed to precisely map outside the X-LAG critical region and definitely did not harbor the X-LAG candidate gene, GPR101. The patients phenotype resembled that of other patients with Xq26 CNVs. The case is instructive for the need for high definition MCs when CG MCs results are inconsistent with the patients phenotype. It is also useful for further supporting the contention that GPR101 is the gene responsible for X-LAG.