Damaging Mutations in AFDN Contribute to Risk of Nonsyndromic Cleft Lip With or Without Cleft Palate.

Novel or rare damaging mutations have been implicated in the developmental pathogenesis of nonsyndromic cleft lip with or without cleft palate (nsCL ± P). Thus, we investigated the human genome for high-impact mutations that could explain the risk of nsCL ± P in our cohorts.We conducted next-generation sequencing (NGS) analysis of 130 nsCL ± P case-parent African trios to identify pathogenic variants that contribute to the risk of clefting. We replicated this analysis using whole-exome sequence data from a Brazilian nsCL ± P cohort. Computational analyses were then used to predict the mechanism by which these variants could result in increased risks for nsCL ± P.We discovered damaging mutations within the AFDN gene, a cell adhesion molecule (CAMs) that was previously shown to contribute to cleft palate in mice. These mutations include p.Met1164Ile, p.Thr453Asn, p.Pro1638Ala, p.Arg669Gln, p.Ala1717Val, and p.Arg1596His. We also discovered a novel splicing p.Leu1588Leu mutation in this protein. Computational analysis suggests that these amino acid changes affect the interactions with other cleft-associated genes including nectins (PVRL1, PVRL2, PVRL3, and PVRL4) CDH1, CTNNA1, and CTNND1.This is the first report on the contribution of AFDN to the risk for nsCL ± P in humans. AFDN encodes AFADIN, an important CAM that forms calcium-independent complexes with nectins 1 and 4 (encoded by the genes PVRL1 and PVRL4). This discovery shows the power of NGS analysis of multiethnic cleft samples in combination with a computational approach in the understanding of the pathogenesis of nsCL ± P.

Whole-genome sequencing reveals de-novo mutations associated with nonsyndromic cleft lip/palate.

The majority (85%) of nonsyndromic cleft lip with or without cleft palate (nsCL/P) cases occur sporadically, suggesting a role for de novo mutations (DNMs) in the etiology of nsCL/P. To identify high impact protein-altering DNMs that contribute to the risk of nsCL/P, we conducted whole-genome sequencing (WGS) analyses in 130 African case-parent trios (affected probands and unaffected parents). We identified 162 high confidence protein-altering DNMs some of which are based on available evidence, contribute to the risk of nsCL/P. These include novel protein-truncating DNMs in the ACTL6A, ARHGAP10, MINK1, TMEM5 and TTN genes; as well as missense variants in ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TULP4. Many of these protein-altering DNMs were predicted to be pathogenic. Analysis using mouse transcriptomics data showed that some of these genes are expressed during the development of primary and secondary palate. Gene-set enrichment analysis of the protein-altering DNMs identified palatal development and neural crest migration among the few processes that were significantly enriched. These processes are directly involved in the etiopathogenesis of clefting. The analysis of the coding sequence in the WGS data provides more evidence of the opportunity for novel findings in the African genome.

Variant analyses of candidate genes in orofacial clefts in multi-ethnic populations.

OBJECTIVES: Cleft lip with/without cleft palate and cleft palate only is congenital birth defects where the upper lip and/or palate fail to fuse properly during embryonic facial development. Affecting ~1.2/1000 live births worldwide, these orofacial clefts impose significant social and financial burdens on affected individuals and their families. Orofacial clefts have a complex etiology resulting from genetic variants combined with environmental covariates. Recent genome-wide association studies and whole-exome sequencing for orofacial clefts identified significant genetic associations and variants in several genes. Of these, we investigated the role of common/rare variants in SHH, RORA, MRPL53, ACVR1, and GDF11. MATERIALS AND METHODS: We sequenced these five genes in 1255 multi-ethnic cleft lip with/without palate and cleft palate only samples in order to find variants that may provide potential explanations for the missing heritability of orofacial clefts. Rare and novel variants were further analyzed using in silico predictive tools. RESULTS: Ninteen total variants of interest were found, with variant types including stop-gain, missense, synonymous, intronic, and splice-site variants. Of these, 3 novel missense variants were found, one in SHH, one in RORA, and one in GDF11. CONCLUSION: This study provides evidence that variants in SHH, RORA, MRPL53, ACVR1, and GDF11 may contribute to risk of orofacial clefts in various populations.

Replication of GWAS significant loci in a sub-Saharan African Cohort with early childhood caries: a pilot study.

BACKGROUND: Early childhood caries (ECC) is a rapidly progressing form of dental infection and a significant public health problem, especially among socially and economically disadvantaged populations. This study aimed to assess the risk factors for ECC among a cohort of Sub-Saharan African children and to determine the role of genetics in the etiology of ECC. METHODS: A sample of 691 children (338 with ECC, 353 without ECC, age < 6 years) was recruited from schools in Lagos, Nigeria. Socio-demographic, dental services utilization and infant dietary data were obtained with interviewer-administered questionnaire. Oral examination was conducted using the WHO oral health diagnostic criteria. Saliva samples were collected from the children for genetic analysis. Single nucleotide polymorphisms were selected from previous study for genotyping. Genetic association analyses to investigate the role of genetics in the etiology of ECC was done. Bivariate comparisons and Multivariate logistic regression analyses were conducted to assess associations between ECC and predictor variables, p < 0.05. RESULTS: Of the 338 children with ECC, 64 (18.9%) had Severe-Early Childhood Caries (S-ECC). Children aged 48-59 months comprised the highest proportion of subjects with ECC (165; 48.8%) and S-ECC (24; 37.5%) while female subjects had higher dt (3.13 ± 2.56) and dmft values 3.27 ± 2.64. ECC was significantly more prevalent among children who were breastfed at night ≥ 12 months (OR 3.30; CI 0.39, 4.75), those with no previous dental visit (OR 1.71; CI 0.24, 2.77), those who used sweetened pacifiers (OR 1.85; CI 0.91, 3.79) and those who daily consumed sugar-sweetened drinks/snacks (OR 1.35; CI 0.09, 18.51). A suggestive increased risk for ECC (OR 1.26, p = 0. 0.0397) was observed for the genetic variant rs11239282 on chromosome 10. We also observed a suggestive reduced risk for ECC (OR 0.80, p = 0.03) for the rs131777 on chromosome 22. None of the genetic variants were significant after correction for multiple testing (Bonferroni p value p = 0.004). CONCLUSIONS: Prolonged night-time breastfeeding, poor utilization of dental services and daily consumption of sugar were risk factors for ECC. Larger sample size is needed to confirm the results of the genetic analysis and to conduct genome wide studies in order to discover new risk loci for ECC.

Non-random distribution of deleterious mutations in the DNA and protein-binding domains of IRF6 are associated with Van Der Woude syndrome.

BACKGROUND: The development of the face occurs during the early days of intrauterine life by the formation of facial processes from the first Pharyngeal arch. Derangement in these well-organized fusion events results in Orofacial clefts (OFC). Van der Woude syndrome (VWS) is one of the most common causes of syndromic cleft lip and/or palate accounting for 2% of all cases. Mutations in the IRF6 gene account for 70% of cases with the majority of these mutations located in the DNA-binding (exon 3, 4) or protein-binding domains (exon 7-9). The current study was designed to update the list of IRF6 variants reported for VWS by compiling all the published mutations from 2013 to date as well as including the previously unreported VWS cases from Africa and Puerto Rico. METHODS: We used PubMed with the search terms; "Van der Woude syndrome," "Popliteal pterygium syndrome," "IRF6," and "Orofacial cleft" to identify eligible studies. We compiled the CADD score for all the mutations to determine the percentage of deleterious variants. RESULTS: Twenty-one new mutations were identified from nine papers. The majority of these mutations were in exon 4. Mutations in exon 3 and 4 had CADD scores between 20 and 30 and mutations in exon 7-9 had CADD scores between 30 and 40. The presence of higher CADD scores in the protein-binding domain (exon 7-9) further confirms the crucial role played by this domain in the function of IRF6. In the new cases, we identified five IRF6 mutations, three novel missense mutations (p.Phe36Tyr, p.Lys109Thr, and p.Gln438Leu), and two previously reported nonsense mutations (p.Ser424*and p.Arg250*). CONCLUSION: Mutations in the protein and DNA-binding domains of IRF6 ranked among the top 0.1% and 1% most deleterious genetic mutations, respectively. Overall, these findings expand the range of VWS mutations and are important for diagnostic and counseling purposes.

Accuracy of unguided and ultrasound guided Coracohumeral ligament infiltrations - a feasibility cadaveric case series.

BACKGROUND: Coracohumeral ligament (CHL) thickening, contracture, and fibroplasia have been identified in glenohumeral idiopathic adhesive capsulitis (GHIAC). The CHL is the main structure responsible for the range of motion limitations. Favorable outcomes have been reported with CHL surgical release. Intra-articular glenohumeral joint corticosteroid infiltrations are utilized to disrupt the inflammatory process and reduce pain in GHIAC. The aim of this study was to investigate whether the CHL could be accurately targeted with a periligamentous infiltration. METHODS: A convenience sample of 12 unembalmed cadaver shoulders (mean age: 74.5 years, range 66-87 years) without evidence of previous injury or surgery were utilized in this exploratory double factor feasibility cadaveric (unguided and ultrasound (US) guided) case series. Two clinicians trained in musculoskeletal infiltration techniques carried out the infiltrations on each shoulder with colored latex. One clinician infiltrated without guidance, the other with US-guidance. The injecting clinicians were blinded to the others infiltration procedure and the order was randomized. An anatomist blinded to the infiltration order performed a shoulder dissection and recorded the infiltrate location. Percentage calculation for accuracy of infiltration and a chi-square evaluation of the difference between unguided and US-guided infiltrations was applied. RESULTS: An accuracy of 75% was achieved for unguided infiltration and 80% for US-guided infiltration techniques. Chi-squared indicated there was no significant difference (p = 0.82) between the unguided and US-guided techniques. CONCLUSION: US-guided and unguided infiltrations achieved good accuracy targeting the CHL, suggesting infiltrations can specifically and accurately target the CHL. In vivo investigation using such infiltration techniques are warranted.

Accuracy of Two Ultrasound-Guided Coracohumeral Ligament Injection Approaches: A Cadaveric Study.

BACKGROUND: Glenohumeral idiopathic adhesive capsulitis is a common shoulder condition that hinders functionality. Addressing the pathology has been extensively researched. Ultrasound (US)-guided injections have shown their efficacy. However, no study has been conducted to compare anatomical accuracy between different approaches in targeting the coracohumeral ligament (CHL). OBJECTIVE: To investigate whether US-guided injection of the CHL can be performed accurately using either the rotator interval (RI) or the coracoidal (CO) approach. METHODS: An experimental cadaveric case series. SETTING: Anatomy laboratory. SPECIMENS: Both shoulders of 13 Thiel-embalmed cadavers. INTERVENTIONS: Three physiatrists each injected a 0.1 mL bolus of colored dye in both shoulders of each cadaver using either the RI or the CO approach under US guidance. Each cadaver received a total of six injections (three injections per shoulder). The accuracy of the injection was determined following shoulder dissection by an anatomist. MAIN OUTCOME MEASURE: The accuracy of the US-guided injection of the CHL. RESULTS: The RI approach yielded 36 accurate injections, giving it an accuracy of 100%. With the CO approach two injections were deemed inaccurate yielding an accuracy of 94%. There was no significant difference in accuracy between all operators. CONCLUSIONS: US-guided injection of the CHL can be performed accurately with both the RI and CO approaches. The RI approach was likely to be more accurate.

Can 5 minutes of repetitive prone press-ups and sustained prone press-ups following a period of spinal loading reverse spinal shrinkage?

Objective: The objective of this study is to investigate if sustained and repetitive prone press-ups could reverse decreased spinal height following spinal loading and if there was a correlation between the degree of end range of motion spinal extension and spinal height gains. Design: Pretest-posttest crossover design is used in this study. Setting: Study was carried out in research laboratory. Subjects: Forty-one healthy men and women were included in this study. Intervention: Participants were seated in the stadiometer for 5 min with a 4.5-kg weight placed on each shoulder; the load was removed for 5 min and spinal height was measured using a stadiometer before and after 5 min of repetitive or sustained prone press-ups. Main Measures: Two-by-two repeated-measures ANOVA to identify significant interactions and main effects is used in this study. Significance of α = 0.05. A Pearson correlation coefficient was used to assess the correlation between spinal height changes and spinal extension ROM. Results: Participants 24.1 ± 2.03 years grew using both repetitive (4.85 ± 3.01 mm) and sustained press ups (4.46 ± 2.57 mm). There was no significant interaction between the repetitive versus sustained press-ups and the time before and after each prone press-ups strategy and no main effect for strategy (sustained vs. repetitive press-ups). There was a significant main effect for time (before vs. after press-ups) (F(1,30) = 140.771; p < 0.0001; partial η2 = 0.82). No correlation was found between the degree of end ROM spinal extension and spinal height changes following press-ups strategies. Conclusion: Following periods of spinal loading, both repetitive and sustained press-ups increased spinal height. Such strategies could be used to help recover spinal height and limit the effects of decreased spinal height as a result of activities of daily living.

Increased spinal height using propped slouched sitting postures: Innovative ways to rehydrate intervertebral discs.

BACKGROUND: Upright and slouched sitting are frequently adopted postures associated with increased intradiscal pressure, spinal height loss and intervertebral disc pathology. OBJECTIVES: To examine the effects of two sustained propped slouched sitting (PSS) postures on spinal height after a period of trunk loading. METHODS: Thirty-four participants without a history of low back pain (LBP) were recruited (age 24.4 ± 1.6 years). Subjects sat in (1) PSS without lumbar support and (2) PSS with lumbar support for 10 min, after a period of trunk loading. Spinal height was measured using a stadiometer. RESULTS: Mean spinal height increase during PSS without lumbar support was 2.94 ± 3.63 mm and with lumbar support 4.74 ± 3.07 mm. CONCLUSIONS: Both PSS with and without lumbar support significantly increased spinal height after a period of trunk loading (p < 0.001). Such PSS postures can provide a valuable alternative to upright sitting and may be recommended for recovering spinal height in the working environment following periods of loading.

Genetic variation in OAS1 is a risk factor for initial infection with West Nile virus in man.

West Nile virus (WNV) is a re-emerging pathogen that can cause fatal encephalitis. In mice, susceptibility to WNV has been reported to result from a single point mutation in oas1b, which encodes 2'-5' oligoadenylate synthetase 1b, a member of the type I interferon-regulated OAS gene family involved in viral RNA degradation. In man, the human ortholog of oas1b appears to be OAS1. The 'A' allele at SNP rs10774671 of OAS1 has previously been shown to alter splicing of OAS1 and to be associated with reduced OAS activity in PBMCs. Here we show that the frequency of this hypofunctional allele is increased in both symptomatic and asymptomatic WNV seroconverters (Caucasians from five US centers; total n = 501; OR = 1.6 [95% CI 1.2-2.0], P = 0.0002 in a recessive genetic model). We then directly tested the effect of this SNP on viral replication in a novel ex vivo model of WNV infection in primary human lymphoid tissue. Virus accumulation varied markedly among donors, and was highest for individuals homozygous for the 'A' allele (P<0.0001). Together, these data identify OAS1 SNP rs10774671 as a host genetic risk factor for initial infection with WNV in humans.

Colorado animal-based plague surveillance systems: relationships between targeted animal species and prediction efficacy of areas at risk for humans.

Human plague risks (Yersinia pestis infection) are greatest when epizootics cause high mortality among this bacterium's natural rodent hosts. Therefore, health departments in plague-endemic areas commonly establish animal-based surveillance programs to monitor Y. pestis infection among plague hosts and vectors. The primary objectives of our study were to determine whether passive animal-based plague surveillance samples collected in Colorado from 1991 to 2005 were sampled from high human plague risk areas and whether these samples provided information useful for predicting human plague case locations. By comparing locations of plague-positive animal samples with a previously constructed GIS-based plague risk model, we determined that the majority of plague-positive Gunnison's prairie dogs (100%) and non-prairie dog sciurids (85.82%), and moderately high percentages of sigmodontine rodents (71.4%), domestic cats (69.3%), coyotes (62.9%), and domestic dogs (62.5%) were recovered within 1 km of the nearest area posing high peridomestic risk to humans. In contrast, the majority of white-tailed prairie dog (66.7%), leporid (cottontailed and jack rabbits) (71.4%), and black-tailed prairie dog (93.0%) samples originated more than 1 km from the nearest human risk habitat. Plague-positive animals or their fleas were rarely (one of 19 cases) collected within 2 km of a case exposure site during the 24 months preceding the dates of illness onset for these cases. Low spatial accuracy for identifying epizootic activity prior to human plague cases suggested that other mammalian species or their fleas are likely more important sources of human infection in high plague risk areas. To address this issue, epidemiological observations and multi-locus variable number tandem repeat analyses (MLVA) were used to preliminarily identify chipmunks as an under-sampled, but potentially important, species for human plague risk in Colorado.

Neurocognitive and functional outcomes in persons recovering from West Nile virus illness.

Long-term neurocognitive and functional impairments following West Nile virus (WNV) disease are poorly understood. We assessed quality-of-life indices and neurocognitive performance in a cohort of 54 persons recovering from one of three WNV disease syndromes (fever [WNF], meningitis [WNM], or encephalitis [WNE]) approximately 1.5 years following acute illness. We compared findings between the three syndromic groups; the study cohort and a demographically similar group of 55 controls from a study of chronic fatigue syndrome (CFS); and the study cohort and a 'normative' control population based on cognitive test data. Persistent symptoms, diminished quality of life, and functional impairment were reported by 50% of WNF patients, and 75% each of WNM and WNE patients. Overall, objective neurocognitive performance did not differ significantly between the three syndromic groups, or between the study cohort and the CFS controls or the normative controls. In some neurocognitive subtests, the study cohort scored below the 15th percentile when compared with normative control data. Most persons who returned to independent living following hospitalization for WNV illness had persistent subjective complaints, but had normal cognitive function. However, a minority displayed subtle neurocognitive deficits more than 18 months following acute disease.

Human plague in the southwestern United States, 1957-2004: spatial models of elevated risk of human exposure to Yersinia pestis.

Plague is a rare but highly virulent flea-borne zoonotic disease caused by the Gram-negative bacterium Yersinia pestis Yersin. Identifying areas at high risk of human exposure to the etiological agent of plague could provide a useful tool for targeting limited public health resources and reduce the likelihood of misdiagnosis by raising awareness of the disease. We created logistic regression models to identify landscape features associated with areas where humans have acquired plague from 1957 to 2004 in the four-corners region of the United States (Arizona, Colorado, New Mexico, and Utah), and we extrapolated those models within a geographical information system to predict where plague cases are likely to occur within the southwestern United States disease focus. The probability of an area being classified as high-risk plague habitat increased with elevation up to approximately 2300 m and declined as elevation increased thereafter, and declined with distance from key habitat types (e.g., southern Rocky Mountain piñon--juniper [Pinus edulis Engelm. and Juniperus spp.], Colorado plateau piñon--juniper woodland, Rocky Mountain ponderosa pine (Pinus ponderosa P.& C. Lawson var. scopulorum), and southern Rocky Mountain juniper woodland and savanna). The overall accuracy of the model was >82%. Our most conservative model predicted that 14.4% of the four-corners region represented a high risk of peridomestic exposure to Y. pestis.

Use of BBL CHROMagar MRSA medium for identification of methicillin-resistant Staphylococcus aureus directly from blood cultures.

We evaluated the ability of BBL CHROMagar MRSA medium (Becton Dickinson, Sparks, MD) to identify methicillin-resistant Staphylococcus aureus (MRSA) directly upon subculture from positive blood culture bottles. There were 124 MRSA isolates recovered from blood cultures in the study. BBL CHROMagar MRSA medium was highly sensitive (97.6% [121/124] at 18 to 24 h of incubation and 100% [124/124] at 48 h) and 99.9% specific for identifying MRSA from positive blood cultures.

West Nile Virus-associated flaccid paralysis outcome.

We report 1-year follow-up data from a longitudinal prospective cohort study of patients with West Nile virus-associated paralysis. As in the 4-month follow-up, a variety of recovery patterns were observed, but persistent weakness was frequent. Respiratory involvement was associated with considerable illness and death.

CCR5 deficiency increases risk of symptomatic West Nile virus infection.

West Nile virus (WNV) is a reemerging pathogen that causes fatal encephalitis in several species, including mouse and human. Recently, we showed that the chemokine receptor CCR5 is critical for survival of mice infected with WNV, acting at the level of leukocyte trafficking to the brain. To test whether this receptor is also protective in man, we determined the frequency of CCR5Delta32, a defective CCR5 allele found predominantly in Caucasians, in two independent cohorts of patients, one from Arizona and the other from Colorado, who had laboratory-confirmed, symptomatic WNV infection. The distribution of CCR5Delta32 in a control population of healthy United States Caucasian random blood donors was in Hardy-Weinberg equilibrium and CCR5Delta32 homozygotes represented 1.0% of the total group (n = 1,318). In contrast, CCR5Delta32 homozygotes represented 4.2% of Caucasians in the Arizona cohort (odds ratios [OR] = 4.4 [95% confidence interval [CI], 1.6-11.8], P = 0.0013) and 8.3% of Caucasians in the Colorado cohort (OR = 9.1 [95% CI, 3.4-24.8], P < 0.0001). CCR5Delta32 homozygosity was significantly associated with fatal outcome in the Arizona cohort (OR = 13.2 [95% CI, 1.9-89.9], P = 0.03). We conclude that CCR5 mediates resistance to symptomatic WNV infection. Because CCR5 is also the major HIV coreceptor, these findings have important implications for the safety of CCR5-blocking agents under development for HIV/AIDS.

West Nile virus-associated flaccid paralysis.

The causes and frequency of acute paralysis and respiratory failure with West Nile virus (WNV) infection are incompletely understood. During the summer and fall of 2003, we conducted a prospective, population-based study among residents of a 3-county area in Colorado, United States, with developing WNV-associated paralysis. Thirty-two patients with developing paralysis and acute WNV infection were identified. Causes included a poliomyelitislike syndrome in 27 (84%) patients and a Guillain-Barré-like syndrome in 4 (13%); 1 had brachial plexus involvement alone. The incidence of poliomyelitislike syndrome was 3.7/100,000. Twelve patients (38%), including 1 with Guillain-Barré-like syndrome, had acute respiratory failure that required endotracheal intubation. At 4 months, 3 patients with respiratory failure died, 2 remained intubated, 25 showed various degrees of improvement, and 2 were lost to followup. A poliomyelitislike syndrome likely involving spinal anterior horn cells is the most common mechanism of WNV-associated paralysis and is associated with significant short- and long-term illness and death.