Open-Source Artificial Intelligence System Supports Diagnosis of Mendelian Diseases in Acutely Ill Infants.

Mendelian disorders are prevalent in neonatal and pediatric intensive care units and are a leading cause of morbidity and mortality in these settings. Current diagnostic pipelines that integrate phenotypic and genotypic data are expert-dependent and time-intensive. Artificial intelligence (AI) tools may help address these challenges. Dx29 is an open-source AI tool designed for use by clinicians. It analyzes the patient's phenotype and genotype to generate a ranked differential diagnosis. We used Dx29 to retrospectively analyze 25 acutely ill infants who had been diagnosed with a Mendelian disorder, using a targeted panel of ~5000 genes. For each case, a trio (proband and both parents) file containing gene variant information was analyzed, alongside patient phenotype, which was provided to Dx29 by three approaches: (1) AI extraction from medical records, (2) AI extraction with manual review/editing, and (3) manual entry. We then identified the rank of the correct diagnosis in Dx29's differential diagnosis. With these three approaches, Dx29 ranked the correct diagnosis in the top 10 in 92-96% of cases. These results suggest that non-expert use of Dx29's automated phenotyping and subsequent data analysis may compare favorably to standard workflows utilized by bioinformatics experts to analyze genomic data and diagnose Mendelian diseases.

SON-Related Zhu-Tokita-Takenouchi-Kim Syndrome With Recurrent Hemiplegic Migraine: Putative Role of PRRT2.

Background and Objectives: Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome (OMIM 617140) is a recently identified neurodevelopmental disorder caused by heterozygous loss-of-function (LoF) variants in SON. Because the SON protein functions as an RNA-splicing regulator, it has been shown that some clinical features of ZTTK syndrome can be attributed to abnormal RNA splicing. Several neurologic features have been observed in patients with ZTTK syndrome, including seizure/epilepsy and other EEG abnormalities. However, a relationship between SON LoF in ZTTK syndrome and hemiplegic migraine remains unknown. Methods: We identified a patient with a pathogenic variant in SON who shows typical clinical features of ZTTK syndrome and experienced recurrent episodes of hemiplegic migraine. To define clinical features, brain MRI and EEG during and after episodes of hemiplegic migraine were characterized. To identify molecular mechanisms for this clinical presentation, we investigated the impact of small interfering RNA (siRNA)-mediated SON knockdown on mRNA expression of the CACNA1A, ATP1A2, SCN1A, and PRRT2 genes, known to be associated with hemiplegic migraine, by quantitative RT-PCR. Pre-mRNA splicing of PRRT2 on SON knockdown was further examined by RT-PCR using primers targeting specific exons. Results: Recurrent episodes of hemiplegic migraine in our patient typically followed modest closed head injuries, and recurrent seizures occurred during the most severe of these episodes. Transient hemispheric cortical interstitial edema and asymmetric EEG slowing were identified during episodes. Our siRNA experiments revealed that SON knockdown significantly reduces PRRT2 mRNA levels in U87MG and SH-SY5Y cell lines, although a reduction in CACNA1A, ATP1A2, and SCN1A mRNA expression was not observed. We further identified that SON knockdown leads to failure in intron 2 removal from PRRT2 pre-mRNA, resulting in a premature termination codon that blocks the generation of functionally intact full-length PRRT2. Discussion: This report identifies recurrent hemiplegic migraine as a novel clinical manifestation of ZTTK syndrome, further characterizes this clinical feature, and provides evidence for downregulation of PRRT2 caused by SON LoF as a mechanism causing hemiplegic migraine. Examination of the SON gene may be indicated in individuals with recurrent hemiplegic migraine.

Multiple endocrine neoplasia type 2 (MEN2) and RET specific modifications of the ACMG/AMP variant classification guidelines and impact on the MEN2 RET database.

The Multiple Endocrine Neoplasia type 2 (MEN2) RET proto-oncogene database, originally published in 2008, is a comprehensive repository of all publicly available RET gene variations associated with MEN2 syndromes. The variant-specific genotype/phenotype information, age of earliest reported medullary thyroid carcinoma (MTC) onset, and relevant references with a brief summary of findings are cataloged. The ACMG/AMP 2015 consensus statement on variant classification was modified specifically for MEN2 syndromes and RET variants using ClinGen sequence variant interpretation working group recommendations and ClinGen expert panel manuscripts, as well as manuscripts from the American Thyroid Association Guidelines Task Force on Medullary Thyroid Carcinoma and other MEN2 RET literature. The classifications for the 166 single unique variants in the MEN2 RET database were reanalyzed using the MEN2 RET specifically modified ACMG/AMP classification guidelines (version 1). Applying these guidelines added two new variant classifications to the database (likely benign and likely pathogenic) and resulted in clinically significant classification changes (e.g., from pathogenic to uncertain) in 15.7% (26/166) of the original variants. Of those clinically significant changes, the highest percentage of changes, 46.2% (12/26), were changes from uncertain to benign or likely benign. The modified ACMG/AMP criteria with MEN2 RET specifications will optimize and standardize RET variant classifications.

Survey of Meat Collected from Commercial Broiler Processing Plants Suggests Low Levels of Semicarbazide Can Be Created during Immersion Chilling.

ABSTRACT: Semicarbazide (SEM) is routinely employed as an indicator for the use of nitrofurazone, a banned antimicrobial. The validity of SEM as a nitrofurazone marker has been scrutinized because of other possible sources of the compound. Nonetheless, a U.S. trade partner rejected skin-on chicken thighs because of SEM detection and suspected nitrofurazone use. Because nitrofurazone has been banned in U.S. broiler production since 2003, we hypothesized that incidental de novo SEM formation occurs during broiler processing. To assess this possibility, raw leg quarters were collected from 23 commercial broiler processing plants across the United States and shipped frozen to our laboratory, where liquid chromatography-mass spectrometry was used to quantitatively assess for SEM. Leg quarter samples were collected at four points along the processing line: hot rehang (transfer from the kill line to the evisceration line), prechill (before the chilling process), postchill (immediately following chilling), and at the point of pack. Thigh meat with skin attached was removed from 535 leg quarters and analyzed in triplicate for SEM concentrations. The concentrations ranged from 0 to 2.67 ppb, with 462 (86.4%) of 535 samples below the regulatory decision level of 0.5 ppb of SEM. The 73 samples over the 0.5-ppb limit came from 21 plants; 53 (72.6%) of positive samples were in meat collected after chilling (postchill or point of pack). The difference in both prevalence and concentration of SEM detected before and after chilling was highly significant (P < 0.0001). These data support our hypothesis that SEM detection in raw broiler meat is related to de novo creation of the chemical during processing.

Comprehensive variant calling from whole-genome sequencing identifies a complex inversion that disrupts ZFPM2 in familial congenital diaphragmatic hernia.

BACKGROUND: Genetic disorders contribute to significant morbidity and mortality in critically ill newborns. Despite advances in genome sequencing technologies, a majority of neonatal cases remain unsolved. Complex structural variants (SVs) often elude conventional genome sequencing variant calling pipelines and will explain a portion of these unsolved cases. METHODS: As part of the Utah NeoSeq project, we used a research-based, rapid whole-genome sequencing (WGS) protocol to investigate the genomic etiology for a newborn with a left-sided congenital diaphragmatic hernia (CDH) and cardiac malformations, whose mother also had a history of CDH and atrial septal defect. RESULTS: Using both a novel, alignment-free and traditional alignment-based variant callers, we identified a maternally inherited complex SV on chromosome 8, consisting of an inversion flanked by deletions. This complex inversion, further confirmed using orthogonal molecular techniques, disrupts the ZFPM2 gene, which is associated with both CDH and various congenital heart defects. CONCLUSIONS: Our results demonstrate that complex structural events, which often are unidentifiable or not reported by clinically validated testing procedures, can be discovered and accurately characterized with conventional, short-read sequencing and underscore the utility of WGS as a first-line diagnostic tool.

Assessing clinical education tools for expanded carrier screening.

Expanded carrier screening (ECS) is increasingly offered to a broader population and raises challenges of how to best educate and counsel the volume of screened individuals. For this study, we compared three educational tools (brochure, video and comic) about ECS on knowledge and decision making. A convenience online sample of 151 pregnant women was randomized to one of three groups (Video, n = 42; Comic n = 54; Brochure n = 55). Knowledge scores were significantly higher for the comic group compared to the video or the brochure groups (p < .001). No significant differences in preparation for decision making, decisional conflict, or perceptions of shared decision making were identified between the study groups. This study suggests that a comic about ECS may improve patient attention and retention of information. The use of graphic narratives may enable individuals to better understand medical information in general.

De novo variants in PAK1 lead to intellectual disability with macrocephaly and seizures.

Using trio exome sequencing, we identified de novo heterozygous missense variants in PAK1 in four unrelated individuals with intellectual disability, macrocephaly and seizures. PAK1 encodes the p21-activated kinase, a major driver of neuronal development in humans and other organisms. In normal neurons, PAK1 dimers reside in a trans-inhibited conformation, where each autoinhibitory domain covers the kinase domain of the other monomer. Upon GTPase binding via CDC42 or RAC1, the PAK1 dimers dissociate and become activated. All identified variants are located within or close to the autoinhibitory switch domain that is necessary for trans-inhibition of resting PAK1 dimers. Protein modelling supports a model of reduced ability of regular autoinhibition, suggesting a gain of function mechanism for the identified missense variants. Alleviated dissociation into monomers, autophosphorylation and activation of PAK1 influences the actin dynamics of neurite outgrowth. Based on our clinical and genetic data, as well as the role of PAK1 in brain development, we suggest that gain of function pathogenic de novo missense variants in PAK1 lead to moderate-to-severe intellectual disability, macrocephaly caused by the presence of megalencephaly and ventriculomegaly, (febrile) seizures and autism-like behaviour.

Targeted gene panel sequencing for the rapid diagnosis of acutely ill infants.

BACKGROUND: Exome/genome sequencing (ES/GS) have been recently used in neonatal and pediatric/cardiac intensive care units (NICU and PICU/CICU) to diagnose and care for acutely ill infants, but the effectiveness of targeted gene panels for these purposes remains unknown. METHODS: RapSeq, a newly developed panel targeting 4,503 disease-causing genes, was employed on selected patients in our NICU/PICU/CICU. Twenty trios were sequenced from October 2015 to March 2017. We assessed diagnostic yield, turnaround times, and clinical consequences. RESULTS: A diagnosis was made in 10/20 neonates (50%); eight had de novo variants (ASXL1, CHD, FBN1, KMT2D, FANCB, FLNA, PAX3), one was a compound heterozygote for CHAT, and one had a maternally inherited GNAS variant. Preliminary reports were generated by 9.6 days (mean); final reports after Sanger sequencing at 16.3 days (mean). In all positive infants, the diagnosis changed management. In a case with congenital myasthenia, diagnosis and treatment occurred at 17 days versus 7 months in a historical control. CONCLUSIONS: This study shows that a gene panel that includes the majority of known disease-causing genes can rapidly identify a diagnosis in a large number of tested infants. Due to simpler deployment and interpretation and lower costs, this approach might represent an alternative to ES/GS in the NICU/PICU/CICU.

Novel mutation in CCBE 1 as a cause of recurrent hydrops fetalis from Hennekam lymphangiectasia-lymphedema syndrome-1.

Whole exome sequencing (WES) was used to determine the etiology of recurrent hydrops fetalis in this case of Hennekam lymphangiectasia-lymphedema syndrome-1. WES is a useful approach for diagnosing rare single-gene conditions with nonspecific phenotypes and should be considered early in the diagnostic process of investigating fetal abnormalities.

The Controlled Palatal Harvest (CPH) Technique for Harvesting a Palatal Subepithelial Connective Tissue Graft.

The use of methamphetamines is a significant public health concern not only in America but worldwide. The consequences of this drug's use are widespread, having major physiologic and psychological effects that are damaging to users' bodies and well-being. Meth use wreaks havoc on one's oral health, with the effects including demineralization of tooth surfaces, carious lesions, inflammation of the periodontium, and more. In addition to examining the neurotoxic effects that methamphetamines have on the brain, this article will discuss how dental professionals can approach treatment of individuals who are affected by this debilitating drug, utilizing a strategy that combines caries arrest with oral hygiene improvement and disease prevention. It will also review the use of various products to help establish a neutral oral pH and enhance enamel remineralization.

Evidence for a role of corticopetal, noradrenergic systems in the development of executive function.

Adolescence is a period during which many aspects of executive function are maturing. Much of the literature has focused on discrepancies between sub-cortical and cortical development that is hypothesized to lead to over-processing of reinforcement related stimuli unchecked by fully matured response inhibition. Specifically, maturation of sub-cortical dopaminergic systems that terminate in the nucleus accumbens has been suggested to occur prior to the full maturation of corticopetal dopaminergic systems. However, converging evidence supports the hypothesis that many aspects of cognitive control are critically linked to cortical noradrenergic systems, that the effectiveness of drugs used to treat disorders of executive function, e.g. ADHD, may result primarily from increases in cortical norepinephrine (NE) and that cortical noradrenergic systems mature across adolescence. However, little attention has been given to the development of this system during adolescence or to its influence in executive function. In the present paper, we discuss the developmental trajectory of the noradrenergic system of the forebrain, highlight the interactions between noradrenergic and dopaminergic systems, and highlight the contribution of the immature corticopetal noradrenergic systems in the ontogeny of several aspects of executive function. Finally we compare data from adolescent rats to those gathered after selective depletion of NE in sub-regions of the prefrontal cortex with an emphasis on the similarities in performance of NE lesioned rats and adolescents.

Reporting incidental findings in genomic scale clinical sequencing--a clinical laboratory perspective: a report of the Association for Molecular Pathology.

Advances in sequencing technologies have facilitated concurrent testing for many disorders, and the results generated may provide information about a patient's health that is unrelated to the clinical indication, commonly referred to as incidental findings. This is a paradigm shift from traditional genetic testing in which testing and reporting are tailored to a patient's specific clinical condition. Clinical laboratories and physicians are wrestling with this increased complexity in genomic testing and reporting of the incidental findings to patients. An enormous amount of discussion has taken place since the release of a set of recommendations from the American College of Medical Genetics and Genomics. This discussion has largely focused on the content of the incidental findings, but the laboratory perspective and patient autonomy have been overlooked. This report by the Association of Molecular Pathology workgroup discusses the pros and cons of next-generation sequencing technology, potential benefits, and harms for reporting of incidental findings, including the effect on both the laboratory and the patient, and compares those with other areas of medicine. The importance of genetic counseling to preserve patient autonomy is also reviewed. The discussion and recommendations presented by the workgroup underline the need for continued research and discussion among all stakeholders to improve our understanding of the effect of different policies on patients, providers, and laboratories.

Genetic counselor review of genetic test orders in a reference laboratory reduces unnecessary testing.

Genetic tests are routinely ordered by health care providers (HCPs) within a wide range of medical specialties. Many providers have limited knowledge or experience with ordering and interpreting genetic tests; thus, test order errors are common. Rigorous review of genetic test orders by genetic counselors (GCs) can provide a direct financial benefit to medical institutions, patients and insurers. GCs at ARUP (Associated Regional University Pathologists) Laboratories routinely perform a preanalytic assessment of complex molecular genetic test orders that includes reviewing clinical and family history information and considering the clinical utility and cost-effectiveness of ordered tests. GCs contact the ordering institution and/or HCP as needed to collect additional clinical information and confirm the test order or suggest alternative testing based on the provided information. A retrospective review of the GC-facilitated test changes over a 21-month period at ARUP laboratories was performed. Approximately 26% of all requests for complex genetic tests assessing germ line mutations were changed following GC review. Testing fees associated with canceled tests were summed to estimate the cost-savings resulting from GC-facilitated test reviews. The test review process resulted in an average reduction in charges to the referring institutions of $48,000.00 per month. GC review of genetic test orders for appropriateness and clinical utility reduces healthcare costs to hospitals, insurers, and patients.

Triplet repeat primed PCR simplifies testing for Huntington disease.

Diagnostic and predictive testing for Huntington disease (HD) requires an accurate determination of the number of CAG repeats in the Huntingtin (HHT) gene. Currently, when a sample appears to be homozygous for a normal allele, additional testing is required to confirm amplification from both alleles. If the sample still appears homozygous, Southern blot analysis is performed to rule out an undetected expanded HTT allele. Southern blot analysis is expensive, time-consuming, and labor intensive and requires high concentrations of DNA. We have developed a chimeric PCR process to help streamline workflow; true homozygous alleles are easily distinguished by this simplified method, and only very large expanded alleles still require Southern blot analysis. Two hundred forty-six HD samples, previously run with a different fragment analysis method, were analyzed with our new method. All samples were correctly genotyped, resulting in 100% concordance between the methods. The chimeric PCR assay was able to identify expanded alleles up to >150 CAG repeats. This method offers a simple strategy to differentiate normal from expanded CAG alleles, thereby reducing the number of samples reflexed to Southern blot analysis. It also provides assurance that expanded alleles are not routinely missed because of allele dropout.

Implementation of a cost-effective unlabeled probe high-resolution melt assay for genotyping of Factor V Leiden.

The Factor V Leiden mutation (FVL; c.1601G>A, p.Arg534Gln), the most common aberration underlying activated Protein C resistance, results in disruption of a major anticoagulation pathway and is a leading cause of inherited thrombophilia. A high-throughput assay for FVL mutation detection was developed using a single unlabeled probe on a high-resolution platform, the 96-well Roche 480 LightCycler (LC480) instrument. This method replaced the U.S. Food and Drug Administration-approved Roche Factor V Leiden kit assay on the LightCycler PCR instrument, decreasing total cost by 48%. The analytical sensitivity and specificity of the LC480 high-resolution assay approached 100% for the FVL mutation. Factor V mutations in proximity to the FVL locus may influence probe binding efficiency and melt characteristics. One out of three very rare variants tested in a separate study, 1600delC, was not distinguishable from FVL using the described high-resolution assay. However, a c.1598G>A variant, which changes the amino acid sequence from arginine to lysine at position 533, was detected by this high-resolution assay and confirmed by bidirectional sequencing. In the labeled probe LightCycler assay, the c.1598G>A variant was indistinguishable from the heterozygous FVL control. The c.1598G>A variant has not been described previously and its clinical significance is uncertain. In conclusion, the LC480 FVL assay is cost effective in a high-throughput setting, with capability to detect both previously described and novel FV variants.

Clinical analysis of PMS2: mutation detection and avoidance of pseudogenes.

Germline mutation detection in PMS2, one of four mismatch repair genes associated with Lynch syndrome, is greatly complicated by the presence of numerous pseudogenes. We used a modification of a long-range PCR method to evaluate PMS2 in 145 clinical samples. This modification avoids potential interference from the pseudogene PMS2CL by utilizing a long-range product spanning exons 11-15, with the forward primer anchored in exon 10, an exon not shared by PMS2CL. Large deletions were identified by MLPA. Pathogenic PMS2 mutations were identified in 22 of 59 patients whose tumors showed isolated loss of PMS2 by immunohistochemistry (IHC), the IHC profile most commonly associated with a germline PMS2 mutation. Three additional patients with pathogenic mutations were identified from 53 samples without IHC data. Thirty-seven percent of the identified mutations were large deletions encompassing one or more exons. In 27 patients whose tumors showed absence of either another protein or combination of proteins, no pathogenic mutations were identified. We conclude that modified long-range PCR can be used to preferentially amplify the PMS2 gene and avoid pseudogene interference, thus providing a clinically useful germline analysis of PMS2. Our data also support the use of IHC screening to direct germline testing of PMS2.

Detection of large rearrangements in the cystic fibrosis transmembrane conductance regulator gene by multiplex ligation-dependent probe amplification assay when sequencing fails to detect two disease-causing mutations.

AIMS: Most of the over 1600 mutations and sequence variants identified to date in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are point mutations or small deletions/insertions detectable by conventional sequencing. However, large rearrangements (deletions, duplications, or insertion/deletion mutations) have recently been reported to constitute 1-2% of CFTR mutations. The CFTR sequencing protocol at ARUP Laboratories interrogates the coding regions of all 27 exons and all intron/exon boundaries of the gene. This study was undertaken to determine whether testing for large gene rearrangements could improve the mutation detection rate. RESULTS: Nine cases with abnormal quantitative pilocarpine iontophoresis sweat chloride (SC) values (>60 mEq/L) and 20 cases with borderline SC levels (40-60 mEq/L) with only one or no mutations detected by the ARUP 32 mutation panel, including the 23 mutations recommended by American College of Medical Genetics (ACMG) for carrier screening, followed by sequencing, were tested using a multiplex ligation-dependent probe amplification (MLPA) assay. MLPA analysis identified one deletion among nine patients with SC >60 who had previously been tested with sequencing. None of the cases with borderline SC levels showed rearrangements. CONCLUSION: The MLPA assay for detection of large rearrangements may be valuable in individuals with positive SC levels where one or no mutations have been identified by sequencing.

Access to care for people with special needs: role of alternative providers and practice settings.

Oral Health in America: A Report of the Surgeon General released in 2000 was the first-ever surgeon general's report on the status of oral health in the United States. It clearly outlined a growing set of challenges in such areas as reducing oral health disparities, improving access to oral and dental care, and prevention of common dental diseases. Findings revealed that 75 percent of dental disease is found in 25 percent of the population. California's children have twice as much untreated decay as their national counterparts. For children with special health care needs seeing a dentist, the data is sparse but a survey of general dentists conducted in 2001 showed that only 10 percent see these children often or very often.