Genetic Testing in Emerging Economies (GenTEE)

Drivers, barriers and opportunities for genetic testing services in emerging economies: the GenTEE (Genetic Testing in Emerging Economies) project Background: Due to the epidemiological transition in the emerging economies of China, East Asia, India, Latin America, the Middle East and South Africa, these economies are facing (i) an increasing proportion of morbidity and mortality due to congenital and genetic conditions, (ii) a rising need for genetic services to improve patient outcomes and overall population health. These economies are facing the challenge how: (i) to ensure the successful translation of genetic/genomics laboratory and academic research into quality assured pathways, (ii) to develop a service delivery infrastructure that leads to equitable and affordable access to high quality genetic/genomic testing services. Objectives: (i) to document and compare current practices and the state of genetic service provision in eight emerging economies: Argentina, Brazil, China, Egypt, India, Oman, Philippines and South Africa, (ii) to identify current knowledge gaps and unmet service needs. The GenTEE international project is intended to inform policy decisions for the challenges of delivering equitable high quality genetic services and to promote international collaboration for capacity building. Methods: (i) a standardized survey that is the first of its worldwide that allows comparison of services internationally across a number of key dimensions by using a core set of indicators, selected by the GenTEE consortium for their relevance and comparability, (ii) capacity building demonstration projects. To date, the GenTEE project has completed its survey that maps the current state of genetic services in the participating countries and identifies current drivers, barriers and opportunities for genetic services development. Results: There is no equitable access to genetic services in all countries mainly due to financial barriers (underfunded fragmented public services, out-of-pocket expenses tend to be the norm for genetic testing services), geographical barriers (concentration of services in main cities) and skill gaps, resulting in inequitable services or delayed access. The development of services in the private sector is opportunistic and mostly technology and market driven. There is a marked lack of standard operating procedures and agreed quality assessment processes for new technologies. Discussion: International collaborative networks can provide support for capacity building and help to strengthen the provision of quality genetic/genomic services in emerging economies.JRC.I.1-Chemical Assessment and Testin

Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development

Fatal Cardiac Arrhythmia and Long-QT Syndrome in a New Form of Congenital Generalized Lipodystrophy with Muscle Rippling (CGL4) Due to PTRF-CAVIN Mutations

We investigated eight families with a novel subtype of congenital generalized lipodystrophy (CGL4) of whom five members had died from sudden cardiac death during their teenage years. ECG studies revealed features of long-QT syndrome, bradycardia, as well as supraventricular and ventricular tachycardias. Further symptoms comprised myopathy with muscle rippling, skeletal as well as smooth-muscle hypertrophy, leading to impaired gastrointestinal motility and hypertrophic pyloric stenosis in some children. Additionally, we found impaired bone formation with osteopenia, osteoporosis, and atlanto-axial instability. Homozygosity mapping located the gene within 2 Mbp on chromosome 17. Prioritization of 74 candidate genes with GeneDistiller for high expression in muscle and adipocytes suggested PTRF-CAVIN (Polymerase I and transcript release factor/Cavin) as the most probable candidate leading to the detection of homozygous mutations (c.160delG, c.362dupT). PTRF-CAVIN is essential for caveolae biogenesis. These cholesterol-rich plasmalemmal vesicles are involved in signal-transduction and vesicular trafficking and reside primarily on adipocytes, myocytes, and osteoblasts. Absence of PTRF-CAVIN did not influence abundance of its binding partner caveolin-1 and caveolin-3. In patient fibroblasts, however, caveolin-1 failed to localize toward the cell surface and electron microscopy revealed reduction of caveolae to less than 3%. Transfection of full-length PTRF-CAVIN reestablished the presence of caveolae. The loss of caveolae was confirmed by Atomic Force Microscopy (AFM) in combination with fluorescent imaging. PTRF-CAVIN deficiency thus presents the phenotypic spectrum caused by a quintessential lack of functional caveolae

Mutations in mitochondrial enzyme GPT2 cause metabolic dysfunction and neurological disease with developmental and progressive features

Mutations that cause neurological phenotypes are highly informative with regard to mechanisms governing human brain function and disease. We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in large kindreds initially ascertained for intellectual and developmental disability (IDD). GPT2 [also known as alanine transaminase 2 (ALT2)] is one of two related transaminases that catalyze the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. In addition to IDD, all affected individuals show postnatal microcephaly and ∼80% of those followed over time show progressive motor symptoms, a spastic paraplegia. Homozygous nonsense p.Arg404* and missense p.Pro272Leu mutations are shown biochemically to be loss of function. The GPT2 gene demonstrates increasing expression in brain in the early postnatal period, and GPT2 protein localizes to mitochondria. Akin to the human phenotype, Gpt2-null mice exhibit reduced brain growth. Through metabolomics and direct isotope tracing experiments, we find a number of metabolic abnormalities associated with loss of Gpt2. These include defects in amino acid metabolism such as low alanine levels and elevated essential amino acids. Also, we find defects in anaplerosis, the metabolic process involved in replenishing TCA cycle intermediates. Finally, mutant brains demonstrate misregulated metabolites in pathways implicated in neuroprotective mechanisms previously associated with neurodegenerative disorders. Overall, our data reveal an important role for the GPT2 enzyme in mitochondrial metabolism with relevance to developmental as well as potentially to neurodegenerative mechanisms.National Institute of Neurological Diseases and Stroke (U.S.) (R01NS035129)United States. National Institutes of Health (R21TW008223)National Cancer Institute (U.S.) (R01CA157996

Developmental and degenerative features in a complicated spastic paraplegia

ObjectiveWe sought to explore the genetic and molecular causes of Troyer syndrome, one of several complicated hereditary spastic paraplegias (HSPs). Troyer syndrome had been thought to be restricted to the Amish; however, we identified 2 Omani families with HSP, short stature, dysarthria and developmental delay—core features of Troyer syndrome—and a novel mutation in the SPG20 gene, which is also mutated in the Amish. In addition, we analyzed SPG20 expression throughout development to infer how disruption of this gene might generate the constellation of developmental and degenerative Troyer syndrome phenotypes.MethodsClinical characterization of 2 non-Amish families with Troyer syndrome was followed by linkage and sequencing analysis. Quantitative polymerase chain reaction and in situ hybridization analysis of SPG20 expression were carried out in embryonic and adult human and mouse tissue.ResultsTwo Omani families carrying a novel SPG20 mutation displayed clinical features remarkably similar to the Amish patients with Troyer syndrome. SPG20 mRNA is expressed broadly but at low relative levels in the adult brain; however, it is robustly and specifically expressed in the limbs, face, and brain during early morphogenesis.InterpretationNull mutations in SPG20 cause Troyer syndrome, a specific clinical entity with developmental and degenerative features. Maximal expression of SPG20 in the limb buds and forebrain during embryogenesis may explain the developmental origin of the skeletal and cognitive defects observed in this disorder. ANN NEUROL 2010;67:516–52

African Head and Neck Society Clinical Practice guidelines for thyroid nodules and cancer in developing countries and limited resource settings

Background International thyroid nodule and cancer management guidelines generally fail to take into account potential limitations in diagnostic and treatment resources. Methods Thyroid cancer specialists from the African Head and Neck Society and American Head & Neck Society Endocrine Section developed guidelines for diagnosis and management of thyroid nodules and cancer in low resource settings. Recommendations were based on literature review and expert opinion, with level of evidence defined. Results Using the ADAPTE process, diagnostic and treatment algorithms were adapted from the National Comprehensive Cancer Network (NCCN). Low resource settings were simulated by systematically removing elements such as availability of laboratory testing, hormone replacement, imaging, and cytopathology from NCCN guidelines. Conclusions Successful management of thyroid nodules and cancer in low resource settings requires adaptation of treatment methodologies. These guidelines define specific scenarios where either more or less aggressive intervention for thyroid pathology may be advisable based on limited available resources

Characterization of greater middle eastern genetic variation for enhanced disease gene discovery

The Greater Middle East (GME) has been a central hub of human migration and population admixture. The tradition of consanguinity, variably practiced in the Persian Gulf region, North Africa, and Central Asia1-3, has resulted in an elevated burden of recessive disease4. Here we generated a whole-exome GME variome from 1,111 unrelated subjects. We detected substantial diversity and admixture in continental and subregional populations, corresponding to several ancient founder populations with little evidence of bottlenecks. Measured consanguinity rates were an order of magnitude above those in other sampled populations, and the GME population exhibited an increased burden of runs of homozygosity (ROHs) but showed no evidence for reduced burden of deleterious variation due to classically theorized ‘genetic purging’. Applying this database to unsolved recessive conditions in the GME population reduced the number of potential disease-causing variants by four- to sevenfold. These results show variegated genetic architecture in GME populations and support future human genetic discoveries in Mendelian and population genetics

Visualization of cell composition and maturation in the bone marrow using 10-color flow cytometry and radar plots

Background: The enormous potential of complex data files generated by 10-color flow cytometry (FC) is hindered by the requirement for exhaustive manual gating and the complexity of multidimensional data visualization. We propose a model using radar plots (RPs), to improve FC data visualization by capturing multidimensionality and integration of FC findings. Method: We analysed 12 normal/reactive bone marrow (N/R BM) samples and 12 BM samples from patients with myelodysplasia (MDS) with 10-color FC. All identifiable cell clusters were individually marked, grouped, and visualized on radar plots. RPs were optimized to de-clutter the cell clusters and map BM cell composition and maturation. Results: A total of 27 immature and mature cell clusters were identified and visualized on 8 multidimensional radar plots. The RPs displayed flow cytometry findings of normal BM in an integrated fashion to maximize overall insight into the data set. The constructed map of bone marrow cell composition was reproducible in all normal BM samples analyzed. Analysis of the pilot cohort of patient samples confirmed the presence of MDS-related changes. These changes are readily identifiable on RPs. Conclusion: We demonstrated that the cell clusters of normal BM can be mapped on multidimensional radar plots, which provide an inclusive insight into BM cell composition and maturation. These reproducible RPs present a comprehensive and comprehensible visual display of differentiation and maturation of haematopoietic cells in normal BM, and can be used as a reference map to assess abnormal haematopoiesis in MDS

Geroderma osteodysplasticum and wrinkly skin syndrome in 22 patients from Oman

Excessive skin wrinkling and cutis laxa are seen in many genetic conditions and overlapping features can make a clinical diagnosis difficult. Here we report on 22 Omani patients from 11 consanguineous families with the diagnosis of wrinkly skin syndrome (WSS, OMIM 278250) or geroderma osteodysplasticum hereditaria (GO, OMIM 231070). The WSS phenotype evolves during early childhood and includes a generalized and excessive skin wrinkling, dental problems, herniae, foot deformities, hip dislocations, growth retardation, and a large anterior fontanelle. The facial gestalt is characterized by a broad nasal bridge, hypertelorism, and downslanting palpebral fissures. We were unable to differentiate between WSS and cutis laxa with growth and developmental delay (CLGDD, OMIM 219200) suggesting that both can be considered as one entity. Distinct hallmarks of GO were skin wrinkling limited to the dorsum of hands and feet and to the abdomen, normal fontanelles, maxillary hypoplasia, bowed long bones, and osteopenia with frequent fractures. In contrast to the attenuation of the skin phenotype with age in WSS, adult patients with GO appeared prematurely aged. A serum sialotransferrin type 2 pattern was found in all four WSS patients tested. Apolipoprotein CIII (a marker for O-glycosylation) was normal suggesting that WSS is frequently associated with a N-protein glycosylation defect, probably at the level of processing (CDG-II). All four investigated GO patients showed normal sialotransferrin patterns. The known loci for cutis laxa and WSS on 2q31, 5q23-q31, 7q11, 11q13, and 14q32 were excluded. We suggest that WSS and GO are distinct entities with overlapping features