Genetic literacy among primary care physicians in a resource-constrained setting.

BACKGROUND: Genetic literacy among primary healthcare providers is crucial for appropriate patient care with the advances in genetic and genomic medicine. Studies from high-income countries highlight the lack of knowledge in genetics and the need to develop curricula for continuing professional development of non-geneticists. Scarce data is available from resource-constrained countries in Middle East and North Africa. Lebanon is a small country in this region characterized by high rates of consanguinity and genetic disorders like several surrounding countries, such as Jordan, Syria, and Turkey. METHODS: The primary aim of this study assessed the genetic literacy, self-perceived and actual knowledge as well as practices among primary care providers in Lebanon. The secondary aim identified their educational needs and proposed evidence-based continuing education programs. A cross-sectional survey-based study, using a self-administered questionnaire, was conducted targeting physicians from Family Medicine, Obstetrics and Gynecology, and Pediatrics. The questionnaire was divided into five sections: demographics, familiarity with genetic tests, self-reported and actual knowledge, genetic practices, and educational needs. Statistics were performed using SPSS v24. The Chi-square test was used for independent variables. Differences between mean scores were measured using paired sample t-tests for groups of two levels and one-way ANOVA for more than two. Multiple linear regression was used to study the variables associated with the knowledge score while controlling for other variables. RESULTS: The survey included 123 physicians. They were mostly familiar with karyotype as first-tier genetic test. Although 38% perceived their knowledge as good, only 6% scored as such in knowledge assessment. A better knowledge score was observed in academic institutions as well as in urban settings (p<0.05). One third never ordered any genetic testing, mostly due to poor knowledge. Almost all (98%) were ready to attend continuing professional development sessions in genetics. CONCLUSION: Our findings show the need to improve genetic literacy among healthcare frontliners, focusing on remote regions and nonacademic centers in Lebanon, a model for other resource-constrained country in the Middle East and North Africa region. This study advances recommendations for evidence-based genetic continuing education programs and highlighted the role of that the few genetic specialists can play in their successful implementation.

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing.

PURPOSE: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains below 50%, suggesting that clinically relevant variants may be missed by standard analyses. Here, we analyze "poison exons" (PEs), which are evolutionarily conserved alternative exons often absent from standard gene annotations. Variants that alter PE inclusion can lead to loss of function and may be highly penetrant contributors to disease. METHODS: We curated published RNA sequencing data from developing mouse cortex to define 1937 conserved PE regions potentially relevant to NDDs, and we analyzed variants found by genome sequencing in multiple NDD cohorts. RESULTS: Across 2999 probands, we found 6 novel clinically relevant variants in PE regions. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family (SCN1A, SCN2A, and SCN8A), which is associated with epilepsies. One variant is in SNRPB, associated with cerebrocostomandibular syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and in genes with gene-phenotype associations consistent with each probands reported features. CONCLUSION: With a very minimal increase in variant analysis burden (average of 0.77 variants per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing.

Purpose: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains around 50%, suggesting some clinically relevant rare variants may be missed by standard analyses. Here we analyze "poison exons" (PEs) which, while often absent from standard gene annotations, are alternative exons whose inclusion results in a premature termination codon. Variants that alter PE inclusion can lead to loss-of-function and may be highly penetrant contributors to disease. Methods: We curated published RNA-seq data from developing mouse cortex to define 1,937 PE regions conserved between humans and mice and potentially relevant to NDDs. We then analyzed variants found by genome sequencing in multiple NDD cohorts. Results: Across 2,999 probands, we found six clinically relevant variants in PE regions that were previously overlooked. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family ( SCN1A, SCN2A , and SCN8A ), associated with epilepsies. One variant is in SNRPB , associated with Cerebrocostomandibular Syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and were observed in probands with features consistent with those reported for the associated gene. Conclusion: With only a minimal increase in variant analysis burden (most probands had zero or one candidate PE variants in a known NDD gene, with an average of 0.77 per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

Reducing Sanger confirmation testing through false positive prediction algorithms.

PURPOSE: Clinical genome sequencing (cGS) followed by orthogonal confirmatory testing is standard practice. While orthogonal testing significantly improves specificity, it also results in increased turnaround time and cost of testing. The purpose of this study is to evaluate machine learning models trained to identify false positive variants in cGS data to reduce the need for orthogonal testing. METHODS: We sequenced five reference human genome samples characterized by the Genome in a Bottle Consortium (GIAB) and compared the results with an established set of variants for each genome referred to as a truth set. We then trained machine learning models to identify variants that were labeled as false positives. RESULTS: After training, the models identified 99.5% of the false positive heterozygous single-nucleotide variants (SNVs) and heterozygous insertions/deletions variants (indels) while reducing confirmatory testing of nonactionable, nonprimary SNVs by 85% and indels by 75%. Employing the algorithm in clinical practice reduced overall orthogonal testing using dideoxynucleotide (Sanger) sequencing by 71%. CONCLUSION: Our results indicate that a low false positive call rate can be maintained while significantly reducing the need for confirmatory testing. The framework that generated our models and results is publicly available at https://github.com/HudsonAlpha/STEVE .

Recommended measures for the efficient care of patients with genetic disorders during the COVID-19 pandemic in low and middle income countries.

The coronavirus disease 2019 (COVID-19) emerged in early 2020 and since, has brought about tremendous cost to economies and healthcare systems universally. Reports of pediatric patients with inherited conditions and COVID-19 infections are emerging. Specific risks for morbidity and mortality that this pandemic carries for different categories of genetic disorders are still mostly unknown. Thus, there are no specific recommendations for the diagnosis, management, and treatment of patients with genetic disorders during the COVID-19 or other pandemics. Emerging publications, from Upper-Middle Income countries (UMIC), discuss the recent experiences of genetic centers in the continuity of care for patients with genetic disorders in the context of this pandemic. Many measures to facilitate the plan to continuous genetic care in a well-developed health system, may not be applicable in Low and Middle Income countries (LMIC). With poorly structured health systems and with the lack of established genetic services, the COVID-19 pandemic will easily exacerbate the access to care for patients with genetic disease in these countries. This article focuses on the unique challenges of providing genetic healthcare services during emergency situations in LMIC countries and provides practical preparations for this and other pandemic situations.

Mapping the Diverse Genetic Disorders and Rare Diseases Among the Syrian Population: Implications on Refugee Health and Health Services in Host Countries.

The aim of this systematic review is to provide physicians and researchers with a comprehensive list of reported genetic disorders in patients of Syrian origin-those who have become part of the largest displaced population globally-and to highlight the need to consider migrant population-based risk for the development of genetic disease control and prevention programs. This review was performed based on the 2015 PRISMA and the international prospective register of systematic reviews. The present review reports on a total of 166 genetic disorders (only 128 reported on OMIM) identified in the Syrian population. Of these disorders, 27% are endocrine-, nutritional- and metabolic-related diseases. Second to metabolic disorders are congenital malformations, deformations and chromosomal abnormalities. Diseases of the blood and the blood-forming organs accounted for 13% of the total genetic disorders. The majority of the genetic disorders reported in Syrian patients followed an autosomal recessive mode of inheritance. These findings are a reflection of the high rates of consanguineous marriages that favor the increase in incidence of these diseases. From the diseases that followed an autosomal recessive mode of inheritance, 22% are reported to be only present in Syria and other regional countries. Twelve of these genetic diseases were identified to be strictly diagnosed in individuals of Syrian origin. The present systematic review highlights the need to develop programs that target genetic disorders affecting Syrian migrants in host countries. These programs would have potential financial and economic benefits, as well as a positive impact on the physical and mental health of members of the Syrian refugee community and those of their host societies. In turn, this would decrease the burden on the health systems in host countries.

A review of the diverse genetic disorders in the Lebanese population: highlighting the urgency for community genetic services.

The review lists the genetic diseases reported in Lebanese individuals, surveys genetic programs and services, and highlights the absence of basic genetic health services at the individual and community level. The incidence of individual diseases is not determined, yet the variety of genetic diseases reported is tremendous, most of which follow autosomal recessive inheritance reflecting the social norms in the population, including high rates of consanguinity, which favor the increase in incidence of these diseases. Genetic services including all activities for the diagnosis, care, and prevention of genetic diseases at community level are extremely inadequate. Services are limited to some clinical and laboratory diagnostic services with no genetic counseling. These services are localized within the capital thus preventing their accessibility to high-risk communities. Screening programs, which are at the core of public health prevention services, are minimal and not nationally mandated. The absence of adequate genetic services is attributed to many factors undermining the importance of genetic diseases and their burden on society, the most important of which is genetic illiteracy at all levels of the population, including high-risk families, the general public, and most importantly health care providers and public health officials. Thus, a country like Lebanon, where genetic diseases are expected to be highly prevalent, is in utmost need for community genetics services. Strategies need to be developed to familiarize public health officials and medical professionals with medical genetics leading to a public health infrastructure that delivers community genetics services for the prevention and care of genetic disorders at community level.

Does dietary folic acid supplementation in mouse NTD models affect neural tube development or gamete preference at fertilization?

BACKGROUND: Neural tube defects (NTDs) are the second most common birth defect in humans. Dietary folic acid (FA) supplementation effectively and safely reduces the incidence of these often debilitating congenital anomalies. FA plays an established role in folate and homocysteine metabolism, but the means by which it suppresses occurrence of NTDs is not understood. In addition, many cases remain resistant to the beneficial effects of folic acid supplementation. To better understand the molecular, biochemical and developmental mechanisms by which FA exerts its effect on NTDs, characterized mouse models are needed that have a defined genetic basis and known response to dietary supplementation. RESULTS: We examined the effect of FA supplementation, at 5-fold the level in the control diet, on the NTD and vertebral phenotypes in Apobtm1Unc and Vangl2Lp mice, hereafter referred to as Apob and Lp respectively. The FA supplemented diet did not reduce the incidence or severity of NTDs in Apob or Lp mutant homozygotes or the loop-tail phenotype in Lp mutant heterozygotes, suggesting that mice with these mutant alleles are resistant to FA supplementation. Folic acid supplementation also did not affect the rate of resorptions or the size of litters, but instead skewed the embryonic genotype distribution in favor of wild-type alleles. CONCLUSION: Similar genotypic biases have been reported for several NTD models, but were interpreted as diet-induced increases in the incidence and severity of NTDs that led to increased embryonic lethality. Absence of differences in resorption rates and litter sizes argue against induced embryonic lethality. We suggest an alternative interpretation, namely that FA supplementation led to strongly skewed allelic inheritance, perhaps from disturbances in polyamine metabolism that biases fertilization in favor of wild-type gametes.

Spectrum of neurological and survival outcomes in pyruvate dehydrogenase complex (PDC) deficiency: lack of correlation with genotype.

Pyruvate dehydrogenase complex (PDC) deficiency is a relatively common mitochondrial disorder that primarily presents with neurological manifestations and lactic acidemia. We analyzed the clinical outcomes and neurological features of 59 consented symptomatic subjects (27 M, 32 F), who were confirmed to have PDC deficiency with defined mutations in one of the genes of PDC (PDHA1, n = 53; PDHB, n = 4; DLAT, n = 2), including 47 different mutations, of which 22 were novel, and for whom clinical records and/or structured interviews were obtained. 39% of these subjects (23/59) have died. Of these, 91% (21/23) died before age 4 years, 61% (14/23) before 1 year, and 43% (10/23) before 3 months. 56% of males died compared with 25% of females. Causes of death included severe lactic acidosis, respiratory failure, and infection. In subjects surviving past 6 months, a broad range of intellectual outcomes was observed. Of 42 subjects whose intellectual abilities were professionally evaluated, 19% had normal or borderline intellectual ability (CQ/IQ ≥ 70), 10% had mild intellectual disability (ID) (CQ/IQ 55-69), 17% had moderate ID (CQ/IQ 40-54), 24% had severe ID (CQ/IQ 25-39) and 33% had profound ID (CQ/IQ<25). Assessment by parents was comparable. Of 10 subjects who reached age 12 years, 9 had had professional IQ assessments, and only 4 had IQs ≥ 70 (only 2 of these 4 had assessments after age 12 years). The average outcome for females was severe-to-profound ID, whereas that of males was mild-to-moderate ID. Of subjects for whom specific neurological data were available, the majority had hypotonia (89%), and hypertonia or mixed hyper-/hypotonia (49%) were common. Seizures (57%), microcephaly (49%), and structural brain abnormalities including ventriculomegaly (67%) and agenesis, dysgenesis, or hypoplasia of the corpus callosum (55%) were common. Leigh syndrome was found in only 35%. Structural brain abnormalities were more common in females, and Leigh syndrome was more common in males. In a subgroup of 16 ambulatory subjects >3.5 years in whom balance was evaluated, ataxia was found in 13. Peripheral neuropathy was documented in 2 cases but not objectively evaluated in most subjects. Outcomes of this population with genetically confirmed PDC deficiency are heterogeneous and not distinctive. Correlations between specific genotypes and outcomes were not established. Although more females survive, related to the prevalence of X-linked PDHA1 mutations, symptomatic surviving females are generally more severely impaired cognitively and have a different pattern of neurological impairment compared to males. Neonatal or infant onset of symptoms was associated with poor outcomes. Males with PDHA1 mutations and low fibroblast PDC activity were less likely to survive beyond infancy. Recurrence rate in siblings of subjects with PDHA1 mutation was less than 5%. Paradoxically, in this retrospective review, potential factors considered possibly relevant to development, such as in vitro PDC activity, specific mutations, use of ketogenic diets, supplements, or medications, were generally not confirmed to be significantly correlated with objective outcomes of survival or neuro-cognitive function. Therefore, the basis of variability of these outcomes remains largely undetermined.

Assays of pyruvate dehydrogenase complex and pyruvate carboxylase activity.

Pyruvate dehydrogenase complex (PDC) and pyruvate carboxylase (PC) are mitochondrial enzymes that provide the initial steps of the two main alternatives for pyruvate metabolism: oxidative decarboxylation vs. anaplerotic carboxylation, gluconeogenesis, and glycerogenesis. Assays of the enzymatic activity of these two enzymes in cells and tissues are described in this chapter, based on evolution or fixation of (14)CO(2). These assays are both suitable for use in crude homogenates of cultured skin fibroblasts, lymphocytes, and frozen muscle (PDC) or liver (PC). Activities of these two enzymes are related to spectrophotometric assays of two other mitochondrial enzymes, dihydrolipoamide dehydrogenase (E3) and citrate synthase (CS), providing initial indices of sample integrity and mitochondrial content. These parameters have proven useful for initial detection of inherited human disorders due to deficiencies of these enzymes, and in combination with available genetic analyses can lead to confirmation of specific diagnoses.

Functional interactions between the LRP6 WNT co-receptor and folate supplementation.

Crooked tail (Cd) mice bear a gain-of-function mutation in Lrp6, a co-receptor for canonical WNT signaling, and are a model of neural tube defects (NTDs), preventable with dietary folic acid (FA) supplementation. Whether the FA response reflects a direct influence of FA on LRP6 function was tested with prenatal supplementation in LRP6-deficient embryos. The enriched FA (10 ppm) diet reduced the occurrence of birth defects among all litters compared with the control (2 ppm FA) diet, but did so by increasing early lethality of Lrp6(-/-) embryos while actually increasing NTDs among nulls alive at embryonic days 10-13 (E10-13). Proliferation in cranial neural folds was reduced in homozygous Lrp6(-/-) mutants versus wild-type embryos at E10, and FA supplementation increased proliferation in wild-type but not mutant neuroepithelia. Canonical WNT activity was reduced in LRP6-deficient midbrain-hindbrain at E9.5, demonstrated in vivo by a TCF/LEF-reporter transgene. FA levels in media modulated the canonical WNT response in NIH3T3 cells, suggesting that although FA was required for optimal WNT signaling, even modest FA elevations attenuated LRP5/6-dependent canonical WNT responses. Gene expression analysis in embryos and adults showed striking interactions between targeted Lrp6 deficiency and FA supplementation, especially for mitochondrial function, folate and methionine metabolism, WNT signaling and cytoskeletal regulation that together implicate relevant signaling and metabolic pathways supporting cell proliferation, morphology and differentiation. We propose that FA supplementation rescues Lrp6(Cd/Cd) fetuses by normalizing hyperactive WNT activity, whereas in LRP6-deficient embryos, added FA further attenuates reduced WNT activity, thereby compromising development.