Clinical utility of targeted next-generation sequencing panel in routine diagnosis of hereditary hemolytic anemia: A national reference laboratory experience.

INTRODUCTION: Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia. MATERIALS AND METHODS: Our hemolytic anemia diagnostic next-generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty-six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019. RESULTS: We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7 TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha-LELY and alpha-LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients. CONCLUSIONS: Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha-LELY and alpha-LEPRA should be included in all targeted HHA panels.

A computational study of structural differences of binding of NADP+ and G6P substrates to G6PD Mediterraneanc.563T, G6PD A-c.202A/c.376G, G6PD Cairoc.404C and G6PD Gazac.536A mutations.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked inherited enzymopathic disorder that may lead to transfusion-requiring acute hemolytic anemia (AHA) triggered by fava beans ingestion, infection or some drugs. The gene encoding for G6PD carries a large number of genetic variants that have varying pathogenicity. We reported on three G6PD variants in the Gaza Strip Palestinian population with differing clinical impacts and frequencies: G6PD Mediterraneanc.563T, African G6PD A-c.202A/c.376G, and G6PD Cairoc.404C. We also identified a novel G6PD missense (Ser179Asn) mutation c.536G > A "G6PD Gaza". In this work we explore the effect of these four genetic variants on the structural and substrate (NADP+ and G6P) binding characteristics of the G6PD enzyme using the Monte Carlo (MC) flexible docking and molecular dynamics (MD) simulation approaches. We report that G6PD A-c.202A/c.376G, G6PD Mediterraneanc.563T, G6PD Cairoc.404C and G6PD Gazac.536A mutations cause significant structural changes in G6PD enzyme to induce conformational instability leading to the loss of binding of one or both substrates and are causative of G6PD deficiency.

Genetic predisposition in type 2 diabetes: A promising approach toward a personalized management of diabetes.

Diabetes mellitus, also known simply as diabetes, has been described as a chronic and complex endocrine metabolic disorder that is a leading cause of death across the globe. It is considered a key public health problem worldwide and one of four important non-communicable diseases prioritized for intervention through world health campaigns by various international foundations. Among its four categories, Type 2 diabetes (T2D) is the commonest form of diabetes accounting for over 90% of worldwide cases. Unlike monogenic inherited disorders that are passed on in a simple pattern, T2D is a multifactorial disease with a complex etiology, where a mixture of genetic and environmental factors are strong candidates for the development of the clinical condition and pathology. The genetic factors are believed to be key predisposing determinants in individual susceptibility to T2D. Therefore, identifying the predisposing genetic variants could be a crucial step in T2D management as it may ameliorate the clinical condition and preclude complications. Through an understanding the unique genetic and environmental factors that influence the development of this chronic disease individuals can benefit from personalized approaches to treatment. We searched the literature published in three electronic databases: PubMed, Scopus and ISI Web of Science for the current status of T2D and its associated genetic risk variants and discus promising approaches toward a personalized management of this chronic, non-communicable disorder.

The CYB5R3c .350C>G and G6PD A alleles modify severity of anemia in malaria and sickle cell disease.

Genetic modifiers of anemia in Plasmodium falciparum infection and sickle cell disease (SCD) are not fully known. Both conditions are associated with oxidative stress, hemolysis and anemia. The CYB5R3 gene encodes cytochrome b5 reductase 3, which converts methemoglobin to hemoglobin through oxidation of NADH. CYB5R3c.350C > G encoding CYB5R3T117S , the most frequent recognized African-specific polymorphism, does not have known functional significance, but its high allele frequency (23% in African Americans) suggests a selection advantage. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection from oxidants; its African-polymorphic X-linked A+ and A- alleles, and other variants with reduced activity, coincide with endemic malaria distribution, suggesting protection from lethal infection. We examined the association of CYB5R3c.350C > G with severe anemia (hemoglobin <5 g/dL) in the context of G6PD A+ and A- status among 165 Zambian children with malaria. CYB5R3c.350C > G offered protection against severe malarial anemia in children without G6PD deficiency (G6PD wild type or A+/A- heterozygotes) (odds ratio 0.29, P = .022) but not in G6PD A+ or A- hemizygotes/homozygotes. We also examined the relationship of CYB5R3c.350C > G with hemoglobin concentration among 267 children and 321 adults and adolescents with SCD in the US and UK and found higher hemoglobin in SCD patients without G6PD deficiency (ß = 0.29, P = .022 children; ß = 0.33, P = .004 adults). Functional studies in SCD erythrocytes revealed mildly lower activity of native CYB5R3T117S compared to wildtype CYB5R3 and higher NADH/NAD+ ratios. In conclusion, CYB5R3c.350C > G appears to ameliorate anemia severity in malaria and SCD patients without G6PD deficiency, possibly accounting for CYB5R3c.350C > G selection and its high prevalence.

Molecular Understanding of Severe Cases of ß-Thalassemia in the Nablus Region, West Bank, Palestine.

Hemoglobinopathies are common inherited monogenic diseases that are likely to remain a serious regional health problem where thalassemias and sickle cell disease are prevalent. In regions where recessive alleles for hemoglobinopathy disorders are present with high consanguinity rates, such as in Palestine, coinheritance of two different genetic defects becomes anticipated and prevalent. In this report, we characterize the molecular variants of the HBB gene for 16 patients with transfusion-dependent anemia registered at the Thalassemia Patient Friends Society in Nablus governorate, West Bank, Palestine. Analysis revealed that 63.0% (10/16) of the patients were homozygous for ß-thalassemia (ß-thal), IVS-I-6 (T>C) (HBB: c.92+6T>C) or IVS-I-110 (G>A) (HBB: c.93-21G>A); 19.0% (3/16) homozygous for sickle cell disease or Hb S (HBB: c.20A>T, p.Glu6Val); 13.0% (2/16) were double heterozygotes for Hb S/ß-thal, (HBB: c.20A>T/HBB: c.92G>C) and HBB: c.20A>T/HBB: c.321_322insG; and one case was a compound heterozygote for ß-thal, codon 39 (C>T) (HBB: c.118C>T) and IVS-I-110. The most common mutation reported in the 16 patients was IVS-I-6 (0.38), followed by IVS-I-110 (0.28) Hb S (0.25) and 0.03 each for codon 39, codons 106/107 (HBB: c.321_322insG) and Hb Monroe (HBB: c.92G>C). In conclusion, in Palestine, a variety of intricate inheritance patterns are encountered in clinical practice.

Loss of Major DNase I Hypersensitive Sites in Duplicated ß-globin Gene Cluster Incompletely Silences HBB Gene Expression.

We report an infant with sickle cell disease phenotype by biochemical analysis whose ß-globin gene (HBB) sequencing showed sickle cell mutation (HBBS ) heterozygosity. The proband has a unique head-to-tail duplication of the ß-globin gene cluster having wild-type (HBBA ) and HBBS alleles inherited from her father; constituting her HBBS /HBBS -HBBA genotype. Further analyses revealed that proband's duplicated ß-globin gene cluster (∼650 kb) encompassing HBBA does not include the immediate upstream locus control region (LCR) or 3' DNase I hypersensitivity (HS) element. The LCR interacts with ß-globin gene cluster involving long range DNA interactions mediated by various transcription factors to drive the regulation of globin genes expression. However, a low level of HBBA transcript was clearly detected by digital PCR. In this patient, the observed transcription from the duplicated, distally displaced HBBA cluster demonstrates that the loss of LCR and flanking 3'HS sites do not lead to complete silencing of HBB transcription.

Favism, the commonest form of severe hemolytic anemia in Palestinian children, varies in severity with three different variants of G6PD deficiency within the same community.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common genetic abnormality known to predispose to acute hemolytic anemia (AHA), which can be triggered by certain drugs or infection. However, the commonest trigger is fava beans (Vicia faba) ingestion, causing AHA (favism), which may be life-threatening especially in children. G6PD deficiency is genetically highly heterogeneous, as nearly 200 different mutations have been observed. We have investigated the hematological features of acute favism in the Palestinian Gaza community that is characterized by the polymorphic coexistence of three different G6PD deficiency genes (G6PD A-, G6PD Cairo, G6PD Med). We have found by comparison to the general population (485 adults and 466 newborns) that children with favism, in terms of relative frequency, G6PD A- was under-represented, whereas G6PD Med was over-represented. We also found that the severity of anemia was significantly greater with G6PD Med and G6PD Cairo than with G6PD A-; and with G6PD Cairo, compared to the other two variants, there was greater hyperbilirubinemia, as well as persistence of mild anemia and reticulocytosis for as long as 4months after recovery from favism. This is the first report determining a differential impact of different G6PD mutations on the clinical features of favism in the same population and the same environment.

Detection of nine Mediterranean ß-thalassemia mutations in Palestinians using three restriction enzyme digest panels: a reliable method for developing countries.

ß-Thalassemia (ß-thal) is a public health problem in the Gaza Strip, Palestine, where about 320 patients are currently managed through blood transfusions and iron chelation. Within the restrictive environment of the Gaza Strip, no advanced molecular analysis [sequencing, real-time polymerase chain reaction (real-time PCR)] technology is currently available for developing a premarital screening protocol and providing couples at risk with prenatal diagnosis. Therefore, genetic identification of samples with indicators of ß-thal is delayed for weeks before the samples can be sequenced outside the country. As nine causative mutations have been identified in the majority of ß-thal cases in the Gaza Strip, a basic genetic screening strategy was designed to improve timeliness in mutation identification and reduce costs to the Palestinian health system. In the present study, we developed a reliable method for the detection of nine Mediterranean ß-thal mutations common to the Palestinian population using a panel of restriction enzyme digests. This strategy utilizes standard instrumentation (thermocycler and agarose gel electrophoresis) that would be available in any basic molecular genetics or biochemical laboratory and provides a reliable method of genetic screening and counseling for patients at risk for ß-thal.

A Single Multiplex PCR and Single-Nucleotide Extension Assay for the Detection of Common Thanatophoric Dysplasia I and II Mutations.

Mutation analysis is used to provide confirmation of a clinical and radiological diagnosis of thanatophoric dysplasia types I and II (TD I and II). We developed a single multiplexed PCR and a single-nucleotide extension (SNE) assay to identify 14 common mutations causing 99% of TD I and TD II, including the challenging three adjacent mutations in the stop codon of exon 18 of the FGFR3 gene. The assay design also provides a solution for resolving SNE PCR product sizing using performance optimized polymer-7. The assay was validated using 37 previously characterized, de-identified patient samples representing the nine wild-types and 10 of 14 mutant genotypes. Four artificial templates were synthesized to mimic four TD I mutations not represented in the available patient samples. Fragment size and fluorophore channel for each SNE product from 10 samples and the four artificial templates were used to define bins and panels for analysis with GeneMarker version 3.0 and GeneMapper version 6.0 software. Allele calls (bin placement within the panels) were verified using the remaining 27 previously characterized samples. This TD I and II PCR and SNE assay is a robust multiplexed assay, streamlined, to identify 14 mutations in one single reaction. Turnaround time required for this assay performance and analysis is decreased in comparison to traditional Sanger or next-generation sequencing.

Unexplained extreme hyperbilirubinemia among neonates in a multihospital healthcare system.

We report a series of neonates who developed a total serum bilirubin (TSB) >20mg/dL during a recent ten-year period in a multihospital healthcare system. The incidence of a TSB >20mg/dL fell after instituting a pre-hospital discharge bilirubin screening program in 2003/2004 (91.3 cases/10,000 births before vs. 72.4/10,000 after), but the incidence has subsequently remained unchanged. No specific cause for the hyperbilirubinemia was identified in 66% of (n=32) cases with a TSB >30 mg/dL or in 76% of (n=112) cases with a TSB 25.0-29.9 mg/dL. We hypothesized that hemolysis was a common contributing mechanism, but our review of hospital records indicated that in most instances these infants were not evaluated sufficiently to test this hypothesis. Records review showed maternal and neonatal blood types and direct antiglobulin testing were performed in >95% cases, but rarely were other tests for hemolysis obtained. In the ten-year period reviewed there were zero instances where erythrocyte morphology from a blood film examination or Heinz body evaluation by a pediatric hematologist or pathologist were performed. In 3% of cases pyruvate kinase was tested, 3% were evaluated by hemoglobin electrophoresis, 3% had a haptoglobin measurement, and 16% were tested for G6PD deficiency. Thus, determining the cause for hyperbilirubinemia in neonates remains a problem at Intermountain Healthcare and, we submit, elsewhere. As a result, the majority of infants with a TSB >25mg/dL have no specific causation identified. We speculate that most of these cases involve hemolysis and that the etiology could be identified if searched for more systematically. With this in mind, we propose a "consistent approach" to evaluating the cause(s) of hyperbilirubinemia among neonates with a TSB >25mg/dL.

"What We Know and What We Do Not Know about Evolutionary Genetic Adaptation to High Altitude Hypoxia in Andean Aymaras".

Three well-studied populations living at high altitudes are Tibetans, Andeans (Aymaras and Quechuas), and Ethiopians. Unlike Tibetans and Ethiopians who have similar hemoglobin (Hb) levels as individuals living at sea level, Aymara Hb levels increase when living at higher altitudes. Our previous whole genome study of Aymara people revealed several selected genes that are involved in cardiovascular functions, but their relationship with Hb levels was not elucidated. Here, we studied the frequencies of known evolutionary-selected variants in Tibetan and Aymara populations and their correlation with high Hb levels in Aymara. We genotyped 177 Aymaras at three different altitudes: 400 m (Santa Cruz), 4000 m (La Paz), and 5000 m (Chorolque), and correlated the results with the elevation of residence. Some of the Tibetan-selected variants also exist in Aymaras, but at a lower prevalence. Two of 10 Tibetan selected variants of EPAS1 were found (rs13005507 and rs142764723) and these variants did not correlate with Hb levels. Allele frequencies of 5 Aymara selected SNPs (heterozygous and homozygous) at 4000 m (rs11578671_BRINP3, rs34913965_NOS2, rs12448902_SH2B1, rs10744822_TBX5, and rs487105_PYGM) were higher compared to Europeans. The allelic frequencies of rs11578671_BRINP3, rs34913965_NOS2, and rs10744822_SH2B1 were significantly higher for Aymaras living at 5000 m than those at 400 m elevation. Variant rs11578671, close to the BRINP3 coding region, correlated with Hb levels in females. Variant rs34913965 (NOS2) correlated with leukocyte counts. Variants rs12448902 (SH2B1) and rs34913965 (NOS2) associated with higher platelet levels. The correlation of these SNPs with blood cell counts demonstrates that the selected genetic variants in Aymara influence hematopoiesis and cardiovascular effects.

Hemolysis and Mediterranean G6PD mutation (c.563 C>T) and c.1311 C>T polymorphism among Palestinians at Gaza Strip.

BACKGROUND: The G6PD c.563 C>T deficient mutation is endemic among Mediterranean populations but its clinical significance is not well delineated. We set up to estimate the proportion of G6PD deficient children presenting with hemolytic anemia at Al Nasser Pediatric Hospital at Gaza Strip, Palestine. We then established the prevalence of c.563T Mediterranean mutation and its linkage to c.1311 C>T polymorphism in this population. DESIGN AND METHODS: G6PD deficiency was identified in children presenting with hemolytic anemia at Al Nasser Pediatric Hospital by spectrophotometric measurement of G6PD activity. G6PD exon 6 and exon 11 were amplified from genomic DNA and evaluated for c.563T mutation by sequencing and the c.1311T polymorphism by restriction fragment analysis. Seventy X-chromosomes (60 males and 5 females) from G6PD deficient patients and 40 X-chromosomes from a control group known to be not G6PD deficient were tested. RESULTS: Over 80% of these children presenting with hemolytic anemia were G6PD deficient and 34% of these had the Mediterranean G6PD deficient variant. The allelic frequencies of Mediterranean c.563T and c.1311T polymorphisms among G6PD deficient patients were 0.33 and 0.38 respectively. The c.1311T polymorphism was linked in 95.2% of patients with the Mediterranean mutation, an allele frequency of 0.87, compared to the control non-G6PD deficient group with an allele frequency of 0.18. CONCLUSIONS: We conclude that G6PD deficiency accounts for majority of hemolytic anemia encountered in Gaza children treated at Al Nasser Pediatric Hospital Emergency department. The Mediterranean mutation c.563T, while not accounting for a majority of G6PD deficiency, is common among G6PD deficient Gaza Strip Palestinians and is frequently, but not always, linked to the c.1311T polymorphism. This work provides a foundation for the population screening of Palestinians for G6PD deficiency and for investigations of ancestral origin of the Mediterranean variant in world populations.

Molecular heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Gaza Strip Palestinians.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting more than 500 million people worldwide, is one of the most common of inherited disorders. There are 186 G6PD mutations published, with mutational clustering within defined ethnic/racial groups. However comprehensive molecular characterization of ethnically associated G6PD mutants and their clinical implications are lacking. DESIGN AND METHODS: Eighty unrelated Palestinian children hospitalized for hemolysis were studied. G6PD activity was determined by quantitative spectrophotometry and G6PD mutations were analyzed by sequencing of gDNA. RESULTS: 65 of 80 children (81%) had G6PD deficiency, accounting for most of the hemolytic disease in this age group. G6PD Mediterranean(c.563T), African G6PD A-(c.202A/c.376G), and G6PD Cairo(c.404C) were common with relative allele frequencies of 0.33 [1], 0.26, and 0.18 respectively. Two other variants were discovered, G6PD Beverly Hills(c.1160A) mutation, and a novel G6PD missense mutation c.536G>A (Ser179Asn), designated G6PD "Gaza". Three samples exhibited enzyme deficiency without detectable exonic or exon/intron boundary mutations. CONCLUSION: G6PD deficiency accounts for the majority of diagnoses for hemolysis in Palestinian children (81%), providing support for newborn G6PD deficiency screening programs. We report unanticipated molecular heterogeneity of G6PD variants among Gaza Strip Palestinians greater than reported in neighboring Arab populations. We report a high proportion of affected children with G6PD Cairo, which was observed previously in only a single Egyptian, and a novel mutation G6PD "Gaza".

A Comprehensive Triple-Repeat Primed PCR and a Long-Range PCR Agarose-Based Assay for Improved Genotyping of Guanine-Adenine-Adenine Repeats in Friedreich Ataxia.

Friedreich ataxia is a rare autosomal recessive, neuromuscular degenerative disease caused by an expansion of a trinucleotide [guanine-adenine-adenine (GAA)] repeat in intron 1 of the FXN gene. It is common in the White population, characterized by progressive gait and limb ataxia, lack of tendon reflexes in the legs, loss of position sense, and hypertrophic cardiomyopathy. Detection and genotyping of the trinucleotide repeat length is important for the diagnosis and prognosis of the disease. A two-tier genotyping assay with an improved triple-repeat primed PCR (TR-PCR) for alleles <200 GAA repeats (±1 to 5 repeats) and an agarose gel-based, long-range PCR (LR-PCR) assay to genotype expanded alleles >200 GAA repeats (±50 repeats) is described. Of the 1236 DNA samples tested using TR-PCR, 31 were identified to have expanded alleles >200 repeats and were reflexed to the LR-PCR procedure for confirmation and quantification. The TR-PCR assay described herein is a diagnostic genotyping assay that reduces the need for further testing. The LR-PCR component is a confirmatory test for true homozygous and heterozygous samples with normal and expanded alleles, as indicated by the TR-PCR assay. The use of this two-tier method offers a comprehensive evaluation to detect and genotype the smallest and largest number of GAA repeats, improving the classification of FXN alleles as normal, mutable normal, borderline, and expanded alleles.

Oral Methylene Blue Treatment in A Dog with Cytochrome B5 Reductase Deficiency And 78, XX Testicular Disorder of Sex Development.

A 6-month-old mixed breed dog was referred for evaluation of a potential disorder of sex development (DSD) and lower than expected energy level. Genitourinary examination revealed ambiguous external genitalia, hypospadias, and a subtle pouch of skin that resembled an empty scrotum. Corrective surgery was planned and subsequently aborted after cyanosis was identified preoperatively and an arterial blood gas analysis by co-oximetry identified increased methemoglobin (MetHb) concentration (35%, normal <2%) with normal arterial oxygen tension. Ensuing investigations confirmed hereditary methemoglobinemia caused by cytochrome b5 reductase (CYB5R) deficiency via molecular genetic (Arg219Pro homozygous variant in CYB5R3 gene) and biochemical (cytochrome b5 reductase enzyme activity of 8% [normal, 100% activity] testing. Karyotyping and molecular analysis of sex chromosomes revealed the dog was genetically female with a normal female karyotype (78,XX), and was negative for the Y-linked SRY gene and positive for the X-linked androgen receptor gene. Methylene blue (MB, 3.3 mg/kg per os [PO] q24 h) was administered and the MetHb concentration decreased to 9% within 14 days. Urogenital revision surgery proceeded without complication and the dog was maintained on MB (3-4 mg/kg PO q24 h) long-term without adverse effects. This is the first report to describe the use of PO MB to decrease MetHb concentrations in a dog with CYB5R deficiency in preparation for anesthesia and highlights its potential as a viable alternative to the intravenous formulation for elective procedures. In addition, this report describes the clinical, molecular, imaging, surgical, and macroscopic and microscopic pathological features of a dog with SRY-negative, 78,XX testicular DSD.

A Novel Variant in G6PD (c.1375C>G) Identified from a Hispanic Neonate with Extreme Hyperbilirubinemia and Low G6PD Enzymatic Activity.

We report a novel glucose-6-phosphate dehydrogenase (G6PD) variant (c.1375C>G) discovered in a 3-day-old Hispanic male child from Salt Lake City, UT, USA. This newborn presented with severe hyperbilirubinemia (29.8 mg/dL or 510 µmol/L) and marked hemolysis evidenced by elevated end-tidal carbon monoxide concentration (5.9 ppm, normal <1.7 ppm). Despite a very low prevalence of G6PD deficiency in Hispanic populations, we pursued testing for this condition and found he had low erythrocyte G6PD enzyme activity (2.8 U/g Hb, normal 9.9-16.6 U/g Hb) and a novel G6PD variant. His mother was heterozygous for this same variant, and she had a moderate decrease in G6PD enzyme activity (7.1 U/g Hb). On the basis of these findings, we propose this variant as a novel pathogenic mutation.