Rapid molecular diagnostics of severe primary immunodeficiency determined by using targeted next-generation sequencing.

BACKGROUND: Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of T cells, B cells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management. OBJECTIVE: We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting. METHODS: The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions. RESULTS: Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7. CONCLUSION: High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life.

Efficacy of erythropoietin in premature infants.

OBJECTIVE: To identify the effect of early parental recombinant human erythropoietin and iron administration on the blood transfusion requirement of premature infants. METHODS: In a controlled clinical trial conducted at the neonatal intensive care unit of Al-Hada Military Hospital, Taif, Kingdom of Saudi Arabia over a 16 months period, we assigned 20 very low birth weight infants with gestational age of (mean +/- standard error of mean) 28.4 +/- 0.5 weeks and birth weight of (mean +/- standard error of mean) 1031 +/- 42 gm, to receive either intravenous recombinant human erythropoietin 200 U/kg/day and iron 1mg/kg/day or conventional therapy over a 21 day study period. Blood transfusion administration undergoes a strict protocol in our nursery. RESULTS: During the 3 week study period, the hemoglobin and hematocrit remained similar in the 2 groups while the reticulocyte counts were greater in the recombinant human erythropoietin recipients on day 14. The number and volume of blood transfusions were similar in both groups. CONCLUSION: Very low birth weight infants receive fewer blood transfusions than the number previously reported. Strict phlebotomy and transfusion criteria could minimize the need for human recombinant erythropoietin.