Comparison between qPCR and RNA-seq reveals challenges of quantifying HLA expression.

Human leukocyte antigen (HLA) class I and II loci are essential elements of innate and acquired immunity. Their functions include antigen presentation to T cells leading to cellular and humoral immune responses, and modulation of NK cells. Their exceptional influence on disease outcome has now been made clear by genome-wide association studies. The exons encoding the peptide-binding groove have been the main focus for determining HLA effects on disease susceptibility/pathogenesis. However, HLA expression levels have also been implicated in disease outcome, adding another dimension to the extreme diversity of HLA that impacts variability in immune responses across individuals. To estimate HLA expression, immunogenetic studies traditionally rely on quantitative PCR (qPCR). Adoption of alternative high-throughput technologies such as RNA-seq has been hampered by technical issues due to the extreme polymorphism at HLA genes. Recently, however, multiple bioinformatic methods have been developed to accurately estimate HLA expression from RNA-seq data. This opens an exciting opportunity to quantify HLA expression in large datasets but also brings questions on whether RNA-seq results are comparable to those by qPCR. In this study, we analyze three classes of expression data for HLA class I genes for a matched set of individuals: (a) RNA-seq, (b) qPCR, and (c) cell surface HLA-C expression. We observed a moderate correlation between expression estimates from qPCR and RNA-seq for HLA-A, -B, and -C (0.2 ≤ rho ≤ 0.53). We discuss technical and biological factors which need to be accounted for when comparing quantifications for different molecular phenotypes or using different techniques.

Global diversity and evidence for coevolution of KIR and HLA.

The killer immunoglobulin-like receptor (KIR) gene cluster shows extensive genetic diversity, as do the HLA class I loci, which encode ligands for KIR molecules. We genotyped 1,642 individuals from 30 geographically distinct populations to examine population-level evidence for coevolution of these two functionally related but unlinked gene clusters. We observed strong negative correlations between the presence of activating KIR genes and their corresponding HLA ligand groups across populations, especially KIR3DS1 and its putative HLA-B Bw4-80I ligands (r = -0.66, P = 0.038). In contrast, we observed weak positive relationships between the various inhibitory KIR genes and their ligands. We observed a negative correlation between distance from East Africa and frequency of activating KIR genes and their corresponding ligands, suggesting a balance between selection on HLA and KIR loci. Most KIR-HLA genetic association studies indicate a primary influence of activating KIR-HLA genotypes in disease risk; concomitantly, activating receptor-ligand pairs in this study show the strongest signature of coevolution of these two complex genetic systems as compared with inhibitory receptor-ligand pairs.

PyPop: a mature open-source software pipeline for population genomics.

Python for Population Genomics (PyPop) is a software package that processes genotype and allele data and performs large-scale population genetic analyses on highly polymorphic multi-locus genotype data. In particular, PyPop tests data conformity to Hardy-Weinberg equilibrium expectations, performs Ewens-Watterson tests for selection, estimates haplotype frequencies, measures linkage disequilibrium, and tests significance. Standardized means of performing these tests is key for contemporary studies of evolutionary biology and population genetics, and these tests are central to genetic studies of disease association as well. Here, we present PyPop 1.0.0, a new major release of the package, which implements new features using the more robust infrastructure of GitHub, and is distributed via the industry-standard Python Package Index. New features include implementation of the asymmetric linkage disequilibrium measures and, of particular interest to the immunogenetics research communities, support for modern nomenclature, including colon-delimited allele names, and improvements to meta-analysis features for aggregating outputs for multiple populations. Code available at: https://zenodo.org/records/10080668 and https://github.com/alexlancaster/pypop.

Killer cell immunoglobulin-like receptor (KIR) gene content variation in the HGDP-CEPH populations.

In the present study, we investigate patterns of variation in the KIR cluster in a large and well-characterized sample of worldwide human populations in the Human Genome Diversity Project-Centre d'Etude du Polymorphisme Humain (HGDP-CEPH) panel in order to better understand the patterns of diversity in the region. Comparison of KIR data with that from other genomic regions allows control for strictly demographic factors; over 500,000 additional genomic markers have been typed in this panel by other investigators and the data made publicly available. Presence/absence frequencies and haplotypic associations for the KIR region are analyzed in the 52 populations comprising the panel and in accordance with major world regions (Africa, Middle East, Central Asia, East Asia, Europe, Americas, and Oceania). These data represent the first overview of KIR population genetics in the well-documented HGDP-CEPH panel and suggest different evolutionary histories and recent selection in the KIR gene cluster.

Improving quality of breast cancer surgery through development of a national breast cancer surgical outcomes (BRCASO) research database.

BACKGROUND: Common measures of surgical quality are 30-day morbidity and mortality, which poorly describe breast cancer surgical quality with extremely low morbidity and mortality rates. Several national quality programs have collected additional surgical quality measures; however, program participation is voluntary and results may not be generalizable to all surgeons. We developed the Breast Cancer Surgical Outcomes (BRCASO) database to capture meaningful breast cancer surgical quality measures among a non-voluntary sample, and study variation in these measures across providers, facilities, and health plans. This paper describes our study protocol, data collection methods, and summarizes the strengths and limitations of these data. METHODS: We included 4524 women ≥18 years diagnosed with breast cancer between 2003-2008. All women with initial breast cancer surgery performed by a surgeon employed at the University of Vermont or three Cancer Research Network (CRN) health plans were eligible for inclusion. From the CRN institutions, we collected electronic administrative data including tumor registry information, Current Procedure Terminology codes for breast cancer surgeries, surgeons, surgical facilities, and patient demographics. We supplemented electronic data with medical record abstraction to collect additional pathology and surgery detail. All data were manually abstracted at the University of Vermont. RESULTS: The CRN institutions pre-filled 30% (22 out of 72) of elements using electronic data. The remaining elements, including detailed pathology margin status and breast and lymph node surgeries, required chart abstraction. The mean age was 61 years (range 20-98 years); 70% of women were diagnosed with invasive ductal carcinoma, 20% with ductal carcinoma in situ, and 10% with invasive lobular carcinoma. CONCLUSIONS: The BRCASO database is one of the largest, multi-site research resources of meaningful breast cancer surgical quality data in the United States. Assembling data from electronic administrative databases and manual chart review balanced efficiency with high-quality, unbiased data collection. Using the BRCASO database, we will evaluate surgical quality measures including mastectomy rates, positive margin rates, and partial mastectomy re-excision rates among a diverse, non-voluntary population of patients, providers, and facilities.

Variability in reexcision following breast conservation surgery.

CONTEXT: Health care reform calls for increasing physician accountability and transparency of outcomes. Partial mastectomy is the most commonly performed procedure for invasive breast cancer and often requires reexcision. Variability in reexcision might be reflective of the quality of care. OBJECTIVE: To assess hospital and surgeon-specific variation in reexcision rates following partial mastectomy. DESIGN, SETTING, AND PATIENTS: An observational study of breast surgery performed between 2003 and 2008 intended to evaluate variability in breast cancer surgical care outcomes and evaluate potential quality measures of breast cancer surgery. Women with invasive breast cancer undergoing partial mastectomy from 4 institutions were studied (1 university hospital [University of Vermont] and 3 large health plans [Kaiser Permanente Colorado, Group Health, and Marshfield Clinic]). Data were obtained from electronic medical records and chart abstraction of surgical, pathology, radiology, and outpatient records, including detailed surgical margin status. Logistic regression including surgeon-level random effects was used to identify predictors of reexcision. MAIN OUTCOME MEASURE: Incidence of reexcision. RESULTS: A total of 2206 women with 2220 invasive breast cancers underwent partial mastectomy and 509 patients (22.9%; 95% CI, 21.2%-24.7%) underwent reexcision (454 patients [89.2%; 95% CI, 86.5%-91.9%] had 1 reexcision, 48 [9.4%; 95% CI, 6.9%-12.0%] had 2 reexcisions, and 7 [1.4%; 95% CI, 0.4%-2.4%] had 3 reexcisions). Among all patients undergoing initial partial mastectomy, total mastectomy was performed in 190 patients (8.5%; 95% CI, 7.2%-9.5%). Reexcision rates for margin status following initial surgery were 85.9% (95% CI, 82.0%-89.8%) for initial positive margins, 47.9% (95% CI, 42.0%-53.9%) for less than 1.0 mm margins, 20.2% (95% CI, 15.3%-25.0%) for 1.0 to 1.9 mm margins, and 6.3% (95% CI, 3.2%-9.3%) for 2.0 to 2.9 mm margins. For patients with negative margins, reexcision rates varied widely among surgeons (range, 0%-70%; P = .003) and institutions (range, 1.7%-20.9%; P < .001). Reexcision rates were not associated with surgeon procedure volume after adjusting for case mix (P = .92). CONCLUSION: Substantial surgeon and institutional variation were observed in reexcision following partial mastectomy in women with invasive breast cancer.

How natural selection shapes genetic differentiation in the MHC region: A case study with Native Americans.

The Human Leukocyte Antigen (HLA) loci are extremely well documented targets of balancing selection, yet few studies have explored how selection affects population differentiation at these loci. In the present study we investigate genetic differentiation at HLA genes by comparing differentiation at microsatellites distributed genomewide to those in the MHC region. Our study uses a sample of 494 individuals from 30 human populations, 28 of which are Native Americans, all of whom were typed for genomewide and MHC region microsatellites. We find greater differentiation in the MHC than in the remainder of the genome (FST-MHC = 0.130 and FST-Genomic = 0.087), and use a permutation approach to show that this difference is statistically significant, and not accounted for by confounding factors. This finding lies in the opposite direction to the expectation that balancing selection reduces population differentiation. We interpret our findings as evidence that selection favors different sets of alleles in distinct localities, leading to increased differentiation. Thus, balancing selection at HLA genes simultaneously increases intra-population polymorphism and inter-population differentiation in Native Americans.

Distribution of killer cell immunoglobulin-like receptors (KIR) and their HLA-C ligands in two Iranian populations.

Killer cell immunoglobulin-like receptors (KIR) gene frequencies vary between populations and contribute to functional variation in immune responses to viruses,autoimmunity and reproductive success. This study describes the frequency distribution of 12 variable KIR genes and their HLA-C ligands in two Iranian populations who have lived for many generations in different environments:t he Azerbaijanis at high altitude and the Jonobi people at sea level. The results are compared with those published for other human populations and a large group of English Caucasians. Differences were seen in KIR and HLA-C group frequencies, in linkage disequilibrium and inhibitory/activating KIR ratios between the groups. Similarities with geographically close populations in the frequencies of the KIR A and B haplotypes and KIR AA genotype reflected their common ancestry. The extreme variability of the KIR gene family and their HLA-C ligands is highlighted and their importance in defining differences between geographically and culturally isolated communities subject to different environmental pressures who come from the same ethnic grouping.

Demographic history and selection at HLA loci in Native Americans.

The American continent was the last to be occupied by modern humans, and native populations bear the marks of recent expansions, bottlenecks, natural selection, and population substructure. Here we investigate how this demographic history has shaped genetic variation at the strongly selected HLA loci. In order to disentangle the relative contributions of selection and demography process, we assembled a dataset with genome-wide microsatellites and HLA-A, -B, -C, and -DRB1 typing data for a set of 424 Native American individuals. We find that demographic history explains a sizeable fraction of HLA variation, both within and among populations. A striking feature of HLA variation in the Americas is the existence of alleles which are present in the continent but either absent or very rare elsewhere in the world. We show that this feature is consistent with demographic history (i.e., the combination of changes in population size associated with bottlenecks and subsequent population expansions). However, signatures of selection at HLA loci are still visible, with significant evidence selection at deeper timescales for most loci and populations, as well as population differentiation at HLA loci exceeding that seen at neutral markers.

Quality assessment of neoadjuvant therapy use in breast conservation: barriers to implementation.

Neoadjuvant systemic therapy (NST) for operable breast cancer can increase the options for conservative surgery in patients with breast cancer. We performed an analysis of a breast cancer outcomes database as a quality assessment of neoadjuvant therapy use in relation to breast conservative rate (BCR). Data were reviewed from a breast cancer database established to monitor outcomes of breast cancer surgery at a tertiary care breast cancer clinic. The frequency of NST-use was correlated to tumor size and BCR. Cause-specific factors for omitting NST in patients undergoing mastectomy for tumors 3 cm or greater were determined. NST was employed in 29 of 241 (12%) cases of invasive breast carcinoma treated surgically from 2003 to 2005. Although a significant decrease in BCR occurred in tumors >3 cm, NST was not frequently employed until tumors reached >5 cm. Defined contraindications to breast conservation (65%) and patient choice for mastectomy (30%) were the two most common reasons for omitting NST in tumors > or = 3 cm. Despite the initial appearance of NST under-utilization in tumors measuring between 3-5 cm, appropriate exclusion of patients not suitable for breast conservation and patient choice for mastectomy both emerged as leading factors for the omission of NST in this group. Use of NST is an important quality metric in optimizing breast conservation. Patient education and greater understanding of patient-related barriers to NST may help improve BCR.

Methods for assessing gene content diversity of KIR with examples from a global set of populations.

A number of statistical methods are widely used to describe allelic variation at specific genetic loci and its implication on the evolutionary history of these loci. Although the methods were developed primarily to study allelic variation at loci that are virtually always present in the genome, they are often applied to data of gene content variation (i.e., presence/absence of multiple homologous genes) at the killer cell immunoglobulin-like receptor (KIR) gene cluster. In this paper, we discuss methodological issues involved in the analysis of gene content variation data in the KIR region and also its covariation with polymorphism at the human leukocyte antigen class I loci, which encode ligands for KIR. A comparison of several statistical methods and measures (gene frequency, haplotype frequency, and linkage disequilibrium estimation) using the Centre d'Etude du Polymorphisme Humain data will be provided using KIR haplotypes that have been determined by segregation analysis, noting the strengths and weaknesses of the methods when only the presence/absence data is considered. Finally, application of these methods to a set of globally distributed populations is described (see Single et al., Nat Genet 39:1114-1119, 2007) in order to illustrate the challenges faced when inferring the joint effects of natural selection and demographic history on these immune-related genes.

Balancing selection and heterogeneity across the classical human leukocyte antigen loci: a meta-analytic review of 497 population studies.

This paper presents a meta-analysis of high-resolution human leukocyte antigen (HLA) allele frequency data describing 497 population samples. Most of the datasets were compiled from studies published in eight journals from 1990 to 2007; additional datasets came from the International Histocompatibility Workshops and from the AlleleFrequencies.net database. In all, these data represent approximately 66,800 individuals from throughout the world, providing an opportunity to observe trends that may not have been evident at the time the data were originally analyzed, especially with regard to the relative importance of balancing selection among the HLA loci. Population genetic measures of allele frequency distributions were summarized across populations by locus and geographic region. A role for balancing selection maintaining much of HLA variation was confirmed. Further, the breadth of this meta-analysis allowed the ranking of the HLA loci, with DQA1 and HLA-C showing the strongest balancing selection and DPB1 being compatible with neutrality. Comparisons of the allelic spectra reported by studies since 1990 indicate that most of the HLA alleles identified since 2000 are very-low-frequency alleles. The literature-based allele-count data, as well as maps summarizing the geographic distributions for each allele, are available online.

Laparoscopic approaches to resection of suspected gastric gastrointestinal stromal tumors based on tumor location.

BACKGROUND: Surgical resection of gastric gastrointestinal stromal tumor (GIST) should be optimized to achieve a negative pathologic surgical margin while limiting the extent of stomach volume loss. Careful identification of exact gastric tumor location using preoperative computed tomography (CT) scans and gastroscopy should allow for selection of a specific operative approach. METHODS: This retrospective case series involved 12 patients (7 men and 5 women; mean age, 60.5 years) with suspected gastric GIST undergoing tumor resection at Fletcher Allen Health Care, a university medical center, from January 2005 to August 2006. The main outcome measures were pathologic resection margins, operative time, estimated blood loss (EBL), morbidity, and duration of hospital stay. RESULTS: The 12 patients were separated into three groups on the basis of tumor location as follows: type 1 (fundus/greater curvature, n = 5), type 2 (prepyloric/antrum, n = 3), and type 3 (lesser curvature/perigastroesophageal junction, n = 4). Preoperative imaging (CT scan and/or endoscopy) used to identify tumor location accurately predicted the operative approach before surgery for 11 of the12 patients. The surgical approach was selected solely by tumor location as follows: type 1 (laparoscopic partial gastrectomy [LPG]), type 2 (laparoscopic distal gastrectomy [LDG]), and type 3 (laparoscopic transgastric resection [LTG]). Nine patients had a final pathologic diagnosis of GIST. The average tumor size was 4.6 cm, but this did not influence procedure selection. Histologic margins were microscopically negative in all patients. The LPG and LTG approaches had similar outcomes in terms of estimated blood loss (EBL; 80 vs 100 ml) and hospital stay (3.4 vs 3.3 days; p = 0.0198), but LTG had longer operative times (236 vs 180 min). The LDG procedure had longer operative times, greater EBL, and a longer hospital stay. The operative morbidity was 17%, and there was no operative mortality. CONCLUSION: The selection of an operative technique for resection of gastric submucosal tumors can be based on preoperative identification of tumor location, for better definition of both the extent of gastric resection and the technical complexity of the laparoscopic procedure.

Signatures of demographic history and natural selection in the human major histocompatibility complex Loci.

Many lines of evidence show that several HLA loci have experienced balancing selection. However, distinguishing among demographic and selective explanations for patterns of variation observed with HLA genes remains a challenge. In this study we address this issue using data from a diverse set of human populations at six classical HLA loci and, employing a comparative genomics approach, contrast results for HLA loci to those for non-HLA markers. Using a variety of analytic methods, we confirm and extend evidence for selection acting on several HLA loci. We find that allele frequency distributions for four of the six HLA loci deviate from neutral expectations and show that this is unlikely to be explained solely by demographic factors. Other features of HLA variation are explained in part by demographic history, including decreased heterozygosity and increased LD for populations at greater distances from Africa and a similar apportionment of genetic variation for HLA loci compared to putatively neutral non-HLA loci. On the basis of contrasts among different HLA loci and between HLA and non-HLA loci, we conclude that HLA loci bear detectable signatures of both natural selection and demographic history.

Enhancing compliance with Medicare guidelines for surgical infection prevention: experience with a cross-disciplinary quality improvement team.

HYPOTHESIS: The Center for Medicare and Medicaid Services instituted standardized reporting of measures aimed at surgical infection prevention (SIP). The complexity and number of medical personnel involved in antibiotic administration requires multiple disciplines to successfully improve compliance. DESIGN: Survey study. SETTING: Tertiary care university hospital. PATIENTS: All patients undergoing the following operations from July 2004 through December 2005 were monitored for compliance with SIP: (1) coronary artery bypass graft, (2) other cardiac, (3) vascular, (4) hysterectomy, (5) colon resection, (6) hip arthroplasty, and (7) knee arthroplasty. INTERVENTION: A team including a surgeon, an anesthesiologist, nurses (preoperative, operating room, and floor), a pharmacist, a hospital infection control committee member, and quality improvement and operations specialists was created in July 2004. Hospital guidelines for SIP were defined, personnel roles defined and processes standardized, and communication/education for health care professionals was enhanced. MAIN OUTCOME MEASURES: Compliance with 3 SIP measures over 3 consecutive periods of 6 months each: (1) percentage of patients receiving antibiotics within 1 hour of incision, (2) percentage of patients with appropriately selected antibiotics, and (3) percentage of patients with antibiotics discontinued within 24 hours of operation end time. RESULTS: One thousand seventy-two patients were monitored. Measure 1 compliance improved from 72.25% to 83.78% (P<.001, Cochran-Armitage trend test); improvement or high performance (>90% compliance) was demonstrated in 5 of 7 services. Measure 2 compliance remained uniformly high (approximately 98%). Measure 3 compliance improved from 54.5% to 87.16% (P<.001); improvement was seen in 5 of 7 services. CONCLUSIONS: The clearly defined roles of a cross-disciplinary team and the process improvements discussed in this article can easily be implemented in other institutions. These elements were integral to our success in improving the timely delivery and discontinuation of prophylactic surgical antibiotics.

Hardy-Weinberg testing of a single homozygous genotype.

No proper statistical test is available for the evaluation of deviation of a single homozygous genotype from Hardy-Weinberg equilibrium (HWE) proportion. We propose a 1-d.f. chi2-test. The power of the proposed test is favorable compared to existing HWE testing procedures. The applications of this test are discussed.

Diversity of MICA and linkage disequilibrium with HLA-B in two North American populations.

The MICA gene has a high degree of polymorphism. Allelic variation of MICA may influence binding of these ligands to the NK cell receptor NKG2D and may affect organ transplantation and/or disease pathogenesis. Knowledge of the population distribution of MICA alleles and their linkage disequilibrium (LD) with class I human leukocyte antigen (HLA) will enhance our understanding of the potential functional significance of the MICA polymorphism. In the present study, we characterized the MICA and HLA-B polymorphisms in two North American populations: European and African. The individual racial groups showed rather limited variation at the MICA locus, where the same set of three most common alleles, MICA*00201, *004, and *00801, account for 64 and 71% of the allele frequency in European-Americans and African-Americans, respectively. Other common alleles (allele frequency >5% in a population) include MICA*00901 and *010. MICA alleles showed strong linkage disequilibrium with HLA-B. Typically, a common MICA allele has strong LD with several HLA-B alleles, whereas most HLA-B alleles and their related serological groups are associated with a single MICA allele. The lack of evidence for an active diversification of the MICA gene after racial separation indicates an evolutionary history distinct from that of the classical HLA genes.

Asymmetric linkage disequilibrium: Tools for assessing multiallelic LD.

Standard measures of linkage disequilibrium (LD) provide an incomplete description of the correlation between two loci. Recently, Thomson and Single (2014) described a new asymmetric pair of LD measures (ALD) that give a more complete description of LD. The ALD measures are symmetric and equivalent to the correlation coefficient r when both loci are bi-allelic. When the numbers of alleles at the two loci differ, the ALD measures capture this asymmetry and provide additional detail about the LD structure. In disease association studies the ALD measures are useful for identifying additional disease genes in a genetic region, by conditioning on known effects. In evolutionary genetic studies ALD measures provide insight into selection acting on individual amino acids of specific genes, or other loci in high LD (see Thomson and Single (2014) for these examples). Here we describe new software for computing and visualizing ALD. We demonstrate the utility of this software using haplotype frequency data from the National Marrow Donor Program (NMDP). This enhances our understanding of LD patterns in the NMDP data by quantifying the degree to which LD is asymmetric and also quantifies this effect for individual alleles.

Development of a quantitative PCR (TaqMan) assay for relative mitochondrial DNA copy number and the common mitochondrial DNA deletion in the rat.

Changes in mitochondrial DNA copy number and increases in mitochondrial DNA mutations, especially deletions, have been associated with exposure to mutagens and with aging. Common deletions that are the result of recombination between direct repeats in human and rat (4,977 and 4,834, bp, respectively) are known to increase in tissues of aged individuals. Previous studies have used long-distance PCR and Southern blot or quantitative PCR to determine the frequency of deleted mitochondrial DNA. A quantitative PCR (TaqMan) assay was developed to detect both mitochondrial DNA copy number and deletion frequency in the rat. This methodology allows not only the determination of changes in the amount of mitochondrial DNA deletion relative to total mitochondrial DNA but also to determine changes in total mitochondrial DNA relative to genomic DNA. As a validation of the assay in rat liver, the frequency of the common 4,834 bp deletion is shown to increase with age, while the relative mitochondrial DNA copy number rises at a young age (3-60 days), then decreases and holds fairly steady to 2 years of age.

Surface EMG electrodes do not accurately record from lumbar multifidus muscles.

OBJECTIVE: This study investigated whether electromyographic signals recorded from the skin surface overlying the multifidus muscles could be used to quantify their activity. DESIGN: Comparison of electromyography signals recorded from electrodes on the back surface and from wire electrodes within four different slips of multifidus muscles of three human subjects performing isometric tasks that loaded the trunk from three different directions. BACKGROUND: It has been suggested that suitably placed surface electrodes can be used to record activity in the deep multifidus muscles. METHODS: We tested whether there was a stronger correlation and more consistent regression relationship between signals from electrodes overlying multifidus and longissimus muscles respectively than between signals from within multifidus and from the skin surface electrodes over multifidus. RESULTS: The findings provided consistent evidence that the surface electrodes placed over multifidus muscles were more sensitive to the adjacent longissimus muscles than to the underlying multifidus muscles. The R(2) for surface versus intra-muscular comparisons was 0.64, while the average R(2) for surface-multifidus versus surface-longissimus comparisons was 0.80. Also, the magnitude of the regression coefficients was less variable between different tasks for the longissimus versus surface multifidus comparisons. CONCLUSIONS: Accurate measurement of multifidus muscle activity requires intra-muscular electrodes. RELEVANCE: Electromyography is the accepted technique to document the level of muscular activation, but its specificity to particular muscles depends on correct electrode placement. For multifidus, intra-muscular electrodes are required.