Biotinidase biochemical and molecular analyses: Experience at a large reference laboratory.

BACKGROUND: Biotinidase deficiency is caused by absent activity of the biotinidase, encoded by the biotinidase gene (BTD). Affected individuals cannot recycle the biotin, leading to heterogeneous symptoms that are primarily neurological and cutaneous. Early treatment with biotin supplementation can prevent irreversible neurological damage and is recommended for patients with profound deficiency, defined as enzyme activity <10% mean normal (MN). Molecular testing has been utilized along with biochemical analysis for diagnosis and management. In this study, our objective was to correlate biochemical phenotype/enzyme activity to BTD genotype in patients for whom both enzyme and molecular testing were performed at our lab, and to review how the correlations inform on variant severity. METHODS: We analyzed results of biotinidase enzyme analysis and BTD gene sequencing in 407 patients where samples were submitted to our laboratory from 2008 to 2020. RESULTS: We identified 84 BTD variants; the most common was c.1330G>C, and 19/84 were novel BTD variants. A total of 36 patients had enzyme activity <10% of MN and the most common variant found in this group was c.528G>T. No variant was reported in one patient in the profound deficiency group. The most common variant found in patients with enzyme activity more than 10% MN was c.1330G>C. CONCLUSIONS: Although enzyme activity alone may be adequate for diagnosing profound biotinidase deficiency, molecular testing is necessary for accurate carrier screening and in cases where the enzyme activity falls in the range where partial deficiency and carrier status cannot be discriminated.

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

Molecular testing for the BRCA1 and BRCA2 Ashkenazi Jewish founder mutations: a report on the College of American Pathologists proficiency testing surveys.

PURPOSE: The purpose of this study was to analyze laboratory performance on proficiency testing surveys offered jointly by the College of American Pathologists/American College of Medical Genetics and Genomics biannually for the three common Ashkenazi Jewish founder mutations in the BRCA1 and BRCA2 genes. METHODS: Survey responses were analyzed for accuracy of genotype determination and the associated clinical interpretation. Data on an individual laboratory's participation over time, number of samples tested, turnaround time, and test methodology were also reviewed. RESULTS: Between 2003 and 2012, 23 US laboratories and 39 international laboratories participated. There were six genotyping errors, with a corresponding analytical sensitivity of 99.0% (479/484 challenges; 95% confidence interval: 97.6-99.7%) and an analytic specificity of 99.9% (870/871; 95% confidence interval: 99.4-99.9%). Among the 1,325 clinical interpretations, 92.5% (1,226/1,325; 95% confidence interval: 91.0-93.9%) matched the intended response. Most of the 99 discrepancies-81% (80/99)-incorrectly interpreted the risk for a negative test result as having a lifetime risk of breast cancer "that is the same as that in the general population" instead of "that cannot be determined without BRCA mutation testing of the affected relative." CONCLUSION: Clinical laboratories demonstrated excellent analytical sensitivity and specificity. The clinical interpretation requires additional education, focusing on the clinical interpretation of negative test results for these three mutations.

Three-year experience of a CAP/ACMG methods-based external proficiency testing program for laboratories offering DNA sequencing for rare inherited disorders.

PURPOSE: Thousands of genetic tests are now offered clinically, but many are for rare disorders that are offered by only a few laboratories. The classic approach to disease-specific external proficiency testing programs is not feasible for such testing, yet calls have been made to provide external oversight. METHODS: A methods-based Sequencing Educational Challenge Survey was launched in 2010, under joint administration of the College of American Pathologists and the American College of Medical Genetics and Genomics. Three sets of Sanger ABI sequence data were distributed twice per year. Participants were asked to identify, formally name, and interpret the sequence variant(s). RESULTS: Between 2010 and 2012, 117 laboratories participated. Using a proposed assessment scheme (e.g., at least 10 of 12 components correct), 98.3% of the 67 US participants had acceptable performance (235 of 239 challenges; 95% confidence interval: 95.8-99.5%) as compared with 88.9% (136 of 153; 95% confidence interval: 82.8-93.4%) for the 50 international participants. CONCLUSION: These data provide a high level of confidence that most US laboratories offering rare disease testing are providing consistent and reliable clinical interpretations. Methods-based proficiency testing programs may be one part of the solution to assessing genetic testing based on next-generation sequencing technology.

Kuskokwim syndrome, a recessive congenital contracture disorder, extends the phenotype of FKBP10 mutations.

Recessive mutations in FKBP10 at 17q21.2, encoding FKBP65, cause both osteogenesis imperfecta (OI) and Bruck syndrome (OI plus congenital contractures). Contractures are a variable manifestation of null/missense FKBP10 mutations. Kuskokwim syndrome (KS) is an autosomal recessive congenital contracture disorder found among Yup'ik Eskimos. Linkage mapping of KS to chromosome 17q21, together with contractures as a feature of FKBP10 mutations, made FKBP10 a candidate gene. We identified a homozygous three-nucleotide deletion in FKBP10 (c.877_879delTAC) in multiple Kuskokwim pedigrees; 3% of regional controls are carriers. The mutation deletes the highly conserved p.Tyr293 residue in FKBP65's third peptidyl-prolyl cis-trans isomerase domain. FKBP10 transcripts are normal, but mutant FKBP65 is destabilized to a residual 5%. Collagen synthesized by KS fibroblasts has substantially decreased hydroxylation of the telopeptide lysine crucial for collagen cross-linking, with 2%-10% hydroxylation in probands versus 60% in controls. Matrix deposited by KS fibroblasts has marked reduction in maturely cross-linked collagen. KS collagen is disorganized in matrix, and fibrils formed in vitro had subtle loosening of monomer packing. Our results imply that FKBP10 mutations affect collagen indirectly, by ablating FKBP65 support for collagen telopeptide hydroxylation by lysyl hydroxylase 2, thus decreasing collagen cross-links in tendon and bone matrix. FKBP10 mutations may also underlie other arthrogryposis syndromes.

NGS-defined measurable residual disease (MRD) after initial chemotherapy as a prognostic biomarker for acute myeloid leukemia.

Treated AML patients often have measurable residual disease (MRD) due to persisting low-level clones. This study assessed whether residual post-treatment somatic mutations, detected by NGS, were significantly prognostic for subsequent clinical outcomes. AML patients (n = 128) underwent both pre-and post-treatment testing with the same 42-gene MRD-validated NGS assay. After induction, 59 (46%) patients were mutation-negative (0.0024 VAF detection limit) and 69 (54%) had ≥1 persisting NGS-detectable mutation. Compared with NGS-negative patients, NGS-positive patients had shorter overall survival (17 months versus median not reached; P = 0.004; hazard ratio = 2.2 [95% CI: 1.3-3.7]) and a shorter time to relapse (14 months versus median not reached; P = 0.014; HR = 1.9 [95% CI: 1.1-3.1]). Among 95 patients with a complete morphologic remission (CR), 43 (45%) were MRD-positive by NGS and 52 (55%) were MRD-negative. These MRD-positive CR patients had a shorter overall survival (16.8 months versus median not reached; P = 0.013; HR = 2.1 [95% CI: 1.2-3.9]) than did the MRD-negative CR patients. Post-treatment persisting MRD positivity, defined by the same NGS-based test used at diagnosis, is thus a more sensitive biomarker for low-level leukemic clones compared to traditional non-molecular methods and is prognostic of subsequent relapse and death.

Brain-derived neurotrophic factor and obesity in the WAGR syndrome.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) has been found to be important in energy homeostasis in animal models, but little is known about its role in energy balance in humans. Heterozygous, variably sized, contiguous gene deletions causing haploinsufficiency of the WT1 and PAX6 genes on chromosome 11p13, approximately 4 Mb centromeric to BDNF (11p14.1), result in the Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome. Hyperphagia and obesity were observed in a subgroup of patients with the WAGR syndrome. We hypothesized that the subphenotype of obesity in the WAGR syndrome is attributable to deletions that induce haploinsufficiency of BDNF. METHODS: We studied the relationship between genotype and body-mass index (BMI) in 33 patients with the WAGR syndrome who were recruited through the International WAGR Syndrome Association. The extent of each deletion was determined with the use of oligonucleotide comparative genomic hybridization. RESULTS: Deletions of chromosome 11p in the patients studied ranged from 1.0 to 26.5 Mb; 58% of the patients had heterozygous BDNF deletions. These patients had significantly higher BMI z scores throughout childhood than did patients with intact BDNF (mean [+/-SD] z score at 8 to 10 years of age, 2.08+/-0.45 in patients with heterozygous BDNF deletions vs. 0.88+/-1.28 in patients without BDNF deletions; P=0.03). By 10 years of age, 100% of the patients with heterozygous BDNF deletions (95% confidence interval [CI], 77 to 100) were obese (BMI > or = 95th percentile for age and sex) as compared with 20% of persons without BDNF deletions (95% CI, 3 to 56; P<0.001). The critical region for childhood-onset obesity in the WAGR syndrome was located within 80 kb of exon 1 of BDNF. Serum BDNF concentrations were approximately 50% lower among the patients with heterozygous BDNF deletions (P=0.001). CONCLUSIONS: Among persons with the WAGR syndrome, BDNF haploinsufficiency is associated with lower levels of serum BDNF and with childhood-onset obesity; thus, BDNF may be important for energy homeostasis in humans.

Arginine to ornithine ratio as a diagnostic marker in patients with positive newborn screening for hyperargininemia.

Arginase deficiency is a rare inborn error of metabolism that interrupts the final step of the urea cycle. Untreated individuals often present with episodic hyperammonemia, developmental delay, cognitive impairment, and spasticity in early childhood. The newborn screening (NBS) algorithms for arginase deficiency vary between individual states in the US but often include hyperargininemia and elevated arginine to ornithine (Arg/Orn) ratio. Here, we report 14 arginase deficiency cases, including two patients with positive NBS for hyperargininemia in whom the diagnosis of arginase deficiency was delayed owing to normal or near normal plasma arginine levels on follow-up testing. To improve the detection capability for arginase deficiency, we evaluated plasma Arg/Orn ratio as a secondary diagnostic marker in positive NBS cases for hyperargininemia. We found that plasma Arg/Orn ratio combined with plasma arginine was a better marker than plasma arginine alone to differentiate patients with arginase deficiency from unaffected newborns. In fact, elevated plasma arginine in combination with an Arg/Orn ratio of ≥1.4 identified all 14 arginase deficiency cases. In addition, we examined the impact of age on plasma arginine and ornithine levels. Plasma arginine increased 0.94 µmol/L/day while ornithine was essentially unchanged in the first 31 days of life, which resulted in a similar increasing trend for the Arg/Orn ratio (0.01/day). This study demonstrated that plasma Arg/Orn ratio as a secondary diagnostic marker improved the detection capability for arginase deficiency in newborns with hyperargininemia, which will allow timely detection of arginase deficiency and hence initiation of treatment before developing symptoms.

Prenatal cell-free DNA screening for fetal aneuploidy in pregnant women at average or high risk: Results from a large US clinical laboratory.

BACKGROUND: We evaluated the performance of a cell-free DNA (cfDNA) prenatal screening assay for trisomies 21, 18, and 13, and sex chromosome aneuploidies (SCAs) among a population of pregnant women that included both those at average and high risk. METHODS: Specimen collection, cfDNA extraction, massively parallel sequencing, and bioinformatics analysis were conducted per laboratory protocol. Assay results, concordance with pregnancy outcomes, and performance characteristics were evaluated. RESULTS: A total 75,658 specimens from 72,176 individual pregnant women were received. Technical reasons accounted for 288 (0.4% of all received samples) tests not performed. In the final analysis cohort (N = 69,794), 13% of pregnancies were considered at average risk and 87% at high risk. Mean gestational age at specimen collection was 15.1 weeks. Of the 69,794 unique pregnancies, 1,359 (1.9%) had positive test results. Among the results with confirmed outcomes, PPV for trisomies 21, 18, and 13 was 98.1%, 88.2%, and 59.3%, respectively; the PPV was 69.0% for SCAs and 75.0% for microdeletions. Overall, PPV was 87.2%, sensitivity was 97.9%, and specificity was 99.9%. CONCLUSION: This cfDNA prenatal screening assay provides highly accurate discrimination between affected and unaffected pregnancies among a population of pregnant women at average and high risk for fetal genetic abnormalities.

Improving Molecular Genetic Test Utilization through Order Restriction, Test Review, and Guidance.

The ordering of molecular genetic tests by health providers not well trained in genetics may have a variety of untoward effects. These include the selection of inappropriate tests, the ordering of panels when the assessment of individual or fewer genes would be more appropriate, inaccurate result interpretation and inappropriate patient guidance, and significant unwarranted cost expenditure. We sought to improve the utilization of molecular genetic tests by requiring providers without specialty training in genetics to use genetic counselors and molecular genetic pathologists to assist in test selection. We used a genetic and genomic test review process wherein the laboratory-based genetic counselor performed the preanalytic assessment of test orders and test triage. Test indication and clinical findings were evaluated against the test panel composition, methods, and test limitations under the supervision of the molecular genetic pathologist. These test utilization management efforts resulted in a decrease in genetic test ordering and a gross cost savings of $1,531,913 since the inception of these programs in September 2011 through December 2013. The combination of limiting the availability of complex genetic tests and providing guidance regarding appropriate test strategies is an effective way to improve genetic tests, contributing to judicious use of limited health care resources.

Verification of performance specifications of a molecular test: cystic fibrosis carrier testing using the Luminex liquid bead array.

CONTEXT: The number of clinical laboratories introducing various molecular tests to their existing test menu is continuously increasing. Prior to offering a US Food and Drug Administration-approved test, it is necessary that performance characteristics of the test, as claimed by the company, are verified before the assay is implemented in a clinical laboratory. OBJECTIVE: To provide an example of the verification of a specific qualitative in vitro diagnostic test: cystic fibrosis carrier testing using the Luminex liquid bead array (Luminex Molecular Diagnostics, Inc, Toronto, Ontario). DESIGN: The approach used by an individual laboratory for verification of a US Food and Drug Administration-approved assay is described. RESULTS: Specific verification data are provided to highlight the stepwise verification approach undertaken by a clinical diagnostic laboratory. CONCLUSIONS: Protocols for verification of in vitro diagnostic assays may vary between laboratories. However, all laboratories must verify several specific performance specifications prior to implementation of such assays for clinical use. We provide an example of an approach used for verifying performance of an assay for cystic fibrosis carrier screening.

Central nervous system embryogenesis and its failures.

The well-orchestrated development of the central nervous system (CNS) requires highly integrated regulatory processes to ensure its precise spatial organization that provides the foundation for proper function. As emphasized in this review, the type, timing, and location of regulatory molecules influence the different stages of development from neuronal induction, regional specification, neuronal specification, and neuronal migration to axonal growth and guidance, neuronal survival, and synapse formation. The known molecular mechanisms are summarized from studies of invertebrates and lower vertebrates, in which we have learned more about the different ligands, receptors, transcription factors, and the intracellular signaling pathways that play specific roles in the different stages of development. Despite known molecular mechanisms of some disturbances, most of the clinical entities that arise from failures of CNS embryogenesis remain unexplained. As more novel genes and their functions are discovered, existing mechanisms will be refined and tenable explanations will be made. With these limitations, two specific clinical entities that have been relatively well studied, holoprosencephaly and neuronal migration defects, are discussed in more detail to illustrate the complexity of regulatory mechanisms that govern well-defined stages of CNS development.