A single center experience with publicly funded clinical exome sequencing for neurodevelopmental disorders or multiple congenital anomalies.

Exome sequencing (ES) is an important diagnostic tool for individuals with neurodevelopmental disorders (NDD) and/or multiple congenital anomalies (MCA). However, the cost of ES limits the test's accessibility for many patients. We evaluated the yield of publicly funded clinical ES, performed at a tertiary center in Israel, over a 3-year period (2018-2020). Probands presented with (1) moderate-to-profound global developmental delay (GDD)/intellectual disability (ID); or (2) mild GDD/ID with epilepsy or congenital anomaly; and/or (3) MCA. Subjects with normal chromosomal microarray analysis who met inclusion criteria were included, totaling 280 consecutive cases. Trio ES (proband and parents) was the default option. In 252 cases (90.0%), indication of NDD was noted. Most probands were males (62.9%), and their mean age at ES submission was 9.3 years (range 1 month to 51 years). Molecular diagnosis was reached in 109 probands (38.9%), mainly due to de novo variants (91/109, 83.5%). Disease-causing variants were identified in 92 genes, 15 of which were implicated in more than a single case. Male sex, families with multiple-affected members and premature birth were significantly associated with lower ES yield (p < 0.05). Other factors, including MCA and coexistence of epilepsy, autism spectrum disorder, microcephaly or abnormal brain magnetic resonance imaging findings, were not associated with the yield. To conclude, our findings support the utility of clinical ES in a real-world setting, as part of a publicly funded genetic workup for individuals with GDD/ID and/or MCA.

Molecular assessment of thymus capabilities in the evaluation of T-cell immunodeficiency.

T-cell immunodeficiency may pose a diagnostic challenge to clinicians, especially when the basic T-cell immune workup is not sufficiently informative. An intensive assessment of thymus capabilities that involves either measuring the recent thymic emigrant cells or analyzing the T-cell receptor (TCR) repertoire is often required to estimate the severity and nature of the immune disorder. A comprehensive T-cell immune workup, including TCR excision circles (TRECs) and TCR repertoire analyses, was performed in three patients with various degrees of severity of T-cell immunodeficiency. All three patients had normal peripheral CD3+ T lymphocytes. TCR repertoire analysis revealed oligoclonal (patient 1), restricted (patient 2), and near-normal (patient 3) patterns. TREC quantification was significantly reduced in patients 1 and 2 but normal in patient 3. Based on clinical features at presentation and at follow-up, and supported by the results of immunologic studies, patients 1 and 2 were diagnosed as having significant T-cell immunodeficiency and patient 3 as having T-cell immunocompetence. Assessment of thymus capabilities by TRECs and TCR repertoire analyses is helpful in diagnosing patients with T-cell immunodeficiency and should be part of the evaluation of every patient suspected of having that condition.

Molecular assessment of thymic capacities in patients with Schimke immuno-osseous dysplasia.

Schimke immuno-osseous dysplasia (SIOD) is caused by SMARCAL1 deficiency and characterized by defective T-cell immunity. The immunodeficiency and the role of thymic function in SIOD patients are not clearly understood. We performed thymic evaluations by assessing T-cell receptor (TCR) diversity, rearrangement, and excision circles in family members with different disease severity carrying the same bi-allelic mutation and in a heterozygous carrier. The expression of SMARCAL1 mRNA in a normal thymic sample was measured using real-time quantitative polymerase chain reaction. Thymus functions were significantly reduced in SIOD patients, and these findings were highly correlated with the clinical phenotype. Quantification of SMARCAL1 mRNA transcript was 3.86-fold higher than normal values for adult kidneys. Genotype alone apparently does not define phenotype, and analysis of TCR diversity, rearrangement, and thymus output can quantify the extent of T-cell immunodeficiency. High thymic expression of SMARCAL1 mRNA raises the possibility of its importance in thymus maintenance and function.

Assessment of the response to imatinib in chronic myeloid leukemia patients--comparison between the FISH, multiplex and RT-PCR methods.

OBJECTIVE: The objective of this study was to evaluate the kinetics of molecular response in chronic myeloid leukemia (CML) patients treated with imatinib and to compare between the fluorescent in situ hybridization (FISH), multiplex and real-time quantitative RT-PCR (RQ-PCR) methods with this respect. METHODS: Molecular follow-up was carried out on 24 CML patients treated with imatinib. FISH analysis was performed according to the standard protocol. For RT-PCR the multiplex and RQ-PCR methods were used. RESULTS: Sixty-three percent and 52% of the patients achieved complete remission according to FISH and multiplex RT-PCR analyses, respectively. Seventy-five percent of the patients achieved remission within the first year of treatment. In 83% of the cases the FISH and RT-PCR results were concordant. RQ-PCR analysis was carried out on 32 of the 41 samples negative by multiplex RT-PCR but only nine were negative. All samples with a BCR-ABL/ABL ratio below 2% were also negative by FISH. There was an excellent correlation between the RQ-PCR and the FISH tests. CONCLUSIONS: Molecular remission according to FISH and multiplex RT-PCR can be achieved by imatinib within 1 yr of therapy. There is a good correlation between the FISH, multiplex and RQ-PCR results in terms of the kinetics of disappearance of the BCR-ABL transcript and the predictability of each method for the other. Although RQ-PCR is the most sensitive method for molecular follow-up, FISH and multiplex RT-PCR can be used as complementary tools, at least during the early period of treatment.