Laboratory testing for preconception/prenatal carrier screening: A technical standard of the American College of Medical Genetics and Genomics (ACMG).

Carrier screening has historically assessed a relatively small number of autosomal recessive and X-linked conditions selected based on frequency in a specific subpopulation and association with severe morbidity or mortality. Advances in genomic technologies enable simultaneous screening of individuals for several conditions. The American College of Medical Genetics and Genomics recently published a clinical practice resource that presents a framework when offering screening for autosomal recessive and X-linked conditions during pregnancy and preconception and recommends a tier-based approach when considering the number of conditions to screen for and their frequency within the US population in general. This laboratory technical standard aims to complement the practice resource and to put forth considerations for clinical laboratories and clinicians who offer preconception/prenatal carrier screening.

Distinctive phenotypes in two children with novel germline RUNX1 mutations - one with myeloid malignancy and increased fetal hemoglobin.

RUNX1 associated familial platelet disorder (FPD) is a rare autosomal dominant hematologic disorder characterized by thrombocytopenia and/or altered platelet function. There is an increased propensity to develop myeloid malignancy (MM) - acute myeloid leukemia, myeloproliferative neoplasms or myelodysplastic syndrome often in association with secondary somatic variants in other genes. To date, 23 FPD-MM pediatric cases have been reported worldwide. Here, we present two new kindreds with novel RUNX1 pathogenic variants in which children are probands. The first family is a daughter/mother diad, sharing a heterozygous frameshift variant in RUNX1 gene (c.501delT p.Ser167Argfs*9). The daughter, age 13 years, presented with features resembling juvenile myelomonocytic leukemia - severe anemia, thrombocytopenia, high white cell count with blast cells, monocytosis, increased nucleated red cells and had somatic mutations with high allele burden in CUX1, PHF6, and SH2B3 genes. She also had increased fetal hemoglobin and increased LIN28B expression. The mother, who had a long history of hypoplastic anemia, had different somatic mutations- a non-coding mutation in CUX1 but none in PHF6 or SH2B3. Her fetal hemoglobin and LIN28B expression were normal. In the second kindred, the proband, now 4 years old with thrombocytopenia alone, was investigated at 3 months of age for persistent neonatal thrombocytopenia with large platelets. Molecular testing identified a heterozygous intragenic deletion in RUNX1 encompassing exon 5. His father is known to have increased bruising for several years but is unavailable for testing. These two cases illustrate the significance of secondary mutations in the development and progression of RUNX1-FPD to MM.

Clonal T-cell large granular lymphocyte proliferations in childhood and young adult immune dysregulation conditions.

BACKGROUND: Proliferation of large granular lymphocytes (LGL) and T-cell LGL (T-LGL) in peripheral blood along with demonstration of clonality are the hallmarks of a heterogeneous group of disorders, including T-LGL leukemia or T-LGL lymphocytosis. They are often associated with neutropenia and responsive to immunosuppression. The true nature of this entity is not well understood. Some cases are reported as reactive phenomena with very limited experience in pediatric population. METHODS: Hematology/Oncology Flow Cytometry Laboratory database has been reviewed retrospectively. Patients with identifiable distinct CD5-dim T-cell population and positive clonal T-cell receptor rearrangement were included in the analysis. Clinical and laboratory data were then reviewed. RESULTS: Sixteen cases of children and young adults with increased peripheral blood clonal T-LGL population characterized by dim CD5 expression with wide range of underlying immune dysregulation/stimulation disorders were reviewed. Extended follow up with repeat testing suggested the reactive nature of persistent clonal T-LGL proliferations in this group. CONCLUSIONS: Our observations indicate that clonal T-LGL proliferations in children and young adults are reactive in nature and some can be persistent with an indolent course with unknown consequentiality. Clonal T-LGL cells could be targeting the most prominent immunogenic stressor(s) involved as a control mechanism.

Molecular classification of endometrial carcinoma applied to endometrial biopsy specimens: Towards early personalized patient management.

BACKGROUND: The current risk stratification systems used to guide management of endometrial cancer are based on irreproducible post surgical pathological information, hence the need for more reliable classification systems. Using microarray and sequencing technologies, TCGA recently identified four prognostically significant endometrial carcinoma subtypes, which subsequently proved reproducible using clinically applicable surrogate tests. Using these tests, we sought to determine the level of concordance between endometrial biopsies and subsequent hysterectomy specimens in assessing the molecular classification of endometrial carcinoma. MATERIALS AND METHODS: Fifty biopsies with corresponding hysterectomy specimens for endometrial carcinomas were collected. Additionally, 10 cases of biopsy proven atypical hyperplasia/EIN who were found to have endometrial carcinoma on resection were included. IHC for mismatch repair (MMR) proteins (MLH1, PMS2, MSH2 and MSH6) and P53 was performed. Microsatellite instability analysis was performed by PCR and Sanger sequencing was performed to detect mutations in exons 9 and 13 of the POLE gene. The level of concordance for tumor grade, histologic subtype, immunohistochemical and molecular profile in both specimens was determined using Cohen's kappa estimates. RESULTS: A high level of concordance was achieved for MMR-loss, MSI-high, P53-wild and abnormal types. In contrast, grade and histologic subtype showed only moderate levels of agreement. POLE gene mutation was detected in two patients. For both cases, mutations were detected only in resection specimens. When comparing atypical hyperplasia/EIN with subsequent hysterectomy tumor, the profile was identical to that of endometrial carcinoma. CONCLUSION: In our cohort of endometrial carcinoma, a high level of concordance was achieved between biopsy and hysterectomy specimens for MMR-loss, MSI-high, P53-wild and abnormal types, superior to that of grade and histologic subtype, providing earlier and more reliable prognostic information to inform management. Similar concordance could not be achieved for POLE mutation, given the low frequency of this mutation in our study.

Acquired Pure Red Cell Aplasia and Acquired Amegakaryocytic Thrombocytopenia Associated With Clonal Expansion of T-Cell Large Granular Lymphocytes in a Patient With Lipopolysaccharide-responsive Beige-like Anchor (LRBA) Protein Deficiency.

Acquired pure red cell aplasia and acquired amegakaryocytic thrombocytopenic purpura are rare in children. Similarly, clonal expansion of T-cell large granular lymphocytes is infrequently seen in pediatrics. Lipopolysaccharide-responsive beige-like anchor (LRBA) protein deficiency is a recently described immunodeficiency syndrome that has been associated with inflammatory bowel disease and autoimmune phenomena such as Evans syndrome. Here, we describe a patient with LRBA deficiency who developed acquired pure red cell aplasia and acquired amegakaryocytic thrombocytopenic purpura associated with expansion of clonal T-cell large granular lymphocytes. This has not been described in the literature previously and adds to the knowledge on the spectrum of manifestations of LRBA deficiency.

EGFR-Mutant Non-Small Cell Lung Cancer in the Era of Precision Medicine: Importance of Germline EGFR T790M Testing.

With the rapid development of precision medicine, next-generation sequencing (NGS) has provided the ability to uncode tumors at the DNA level. Identifying EGFR mutations and other molecular changes has become more crucial in the management of non-small cell lung cancer (NSCLC) than ever before. Although the histologic subtypes in patients with advanced NSCLC remain valid in determining treatment options, the detection of specific molecular signatures such as de novo T790M with sensitizing EGFR mutations could be more useful than the histologic subtype itself. Germline T790M mutation should be suspected and tested for when multiple biopsies show de novo T790M mutations or when de novo T790M is found in patients with a family history of lung cancer. This case report presents a 60-year-old woman with bilateral NSCLC with 3 different distinct histologic diagnoses. Evaluating the molecular profile using NGS completely changed the diagnosis, prognosis, and management of this rare presentation of NSCLC.

Extra alleles in FMR1 triple-primed PCR: artifact, aneuploidy, or somatic mosaicism?

Triple-primed PCR assays have become the preferred fragile X syndrome testing method. Using a commercially available assay, we detected a reproducible extra peak(s) in 0.5% of 13,161 clinical samples. The objectives of this study were to determine the cause of these extra peaks; to identify whether these peaks represent an assay specific artifact, an underlying chromosome aneuploidy, or somatic mosaicism; and to ascertain their clinical relevance. The presence of an extra allele(s) was confirmed by a laboratory-developed PCR, with sequencing of the FMR1 5' UTR or Southern blot for some samples. The laboratory-developed procedure detected the extra allele(s) in 57 of 64 samples. Thus, we confirmed an extra peak, typically of lower abundance, in approximately 0.4% of all samples. Of these samples, 5 were from males and 52 were from heterozygous or homozygous females. Six patients likely had X chromosome aneuploidies. In 82.3% of samples, the extra allele had fewer repeats than the predominant allele(s). Additional alleles detected by FMR1 triple-primed PCR are not an assay-specific artifact and are likely due to X chromosome aneuploidies or somatic repeat instability. Additional normal alleles likely have no clinical significance for fragile X syndrome carrier or affected status. Extra alleles in individuals with normal karyotypes probably represent FMR1 somatic variation.

scAAV9 intracisternal delivery results in efficient gene transfer to the central nervous system of a feline model of motor neuron disease.

On the basis of previous studies suggesting that vascular endothelial growth factor (VEGF) could protect motor neurons from degeneration, adeno-associated virus vectors (serotypes 1 and 9) encoding VEGF (AAV.vegf) were administered in a limb-expression 1 (LIX1)-deficient cat-a large animal model of lower motor neuron disease-using three different delivery routes to the central nervous system. AAV.vegf vectors were injected into the motor cortex via intracerebral administration, into the cisterna magna, or intravenously in young adult cats. Intracerebral injections resulted in detectable transgene DNA and transcripts throughout the spinal cord, confirming anterograde transport of AAV via the corticospinal pathway. However, such strategy led to low levels of VEGF expression in the spinal cord. Similar AAV doses injected intravenously resulted also in poor spinal cord transduction. In contrast, intracisternal delivery of AAV exhibited long-term transduction and high levels of VEGF expression in the entire spinal cord, yet with no detectable therapeutic clinical benefit in LIX1-deficient animals. Altogether, we demonstrate (i) that intracisternal delivery is an effective AAV delivery route resulting in high transduction of the entire spinal cord, associated with little to no off-target gene expression, and (ii) that in a LIX1-deficient cat model, however, VEGF expressed at high levels in the spinal cord has no beneficial impact on the disease course.

Failure of lower motor neuron radial outgrowth precedes retrograde degeneration in a feline model of spinal muscular atrophy.

Feline spinal muscular atrophy (SMA) is a fully penetrant, autosomal recessive lower motor neuron disease in domestic cats that clinically resembles human SMA Type III. A whole genome linkage scan identified a ∼140-kb deletion that abrogates expression of LIX1, a novel SMA candidate gene of unknown function. To characterize the progression of feline SMA, we assessed pathological changes in muscle and spinal cord from 3 days of age to beyond onset of clinical signs. Electromyographic (EMG) analysis indicating denervation occurred between 10 and 12 weeks, with the first neurological signs occurring at the same time. Compound motor action potential (CMAP) amplitudes were significantly reduced in the soleus and extensor carpi radialis muscles at 8-11 weeks. Quadriceps femoris muscle fibers from affected cats appeared smaller at 10 weeks; by 12 weeks atrophic fibers were more prevalent than in age-matched controls. In affected cats, significant loss of L5 ventral root axons was observed at 12 weeks. By 21 weeks of age, affected cats had 40% fewer L5 motor axons than normal. There was no significant difference in total L5 soma number, even at 21 weeks; thus degeneration begins distal to the cell body and proceeds retrogradely. Morphometric analysis of L5 ventral roots and horns revealed that 4 weeks prior to axon loss, motor axons in affected cats failed to undergo radial enlargement, suggesting a role for the putative disease gene LIX1 in radial growth of axons.

Lix1 knockout mouse does not exhibit spinal muscular atrophy phenotype.

Feline spinal muscular atrophy (SMA) is an autosomal recessive juvenile onset lower motor neuron disease caused by an ∼ 140 kb deletion that disrupts expression of 2 genes, limb expression 1 (LIX1) and leucyl/cystinyl aminopeptidase (LNPEP). A previously generated Lnpep knockout (KO) mouse did not demonstrate a neuromuscular phenotype. Little is known about LIX1, except that it is evolutionarily conserved and highly expressed in spinal cord motor neurons. To determine whether loss of LIX1 alone is responsible for the feline SMA phenotype, a Lix1 intron 1 gene trap KO mouse line was obtained from Lexicon Genetics, Inc. Mating of F(1) heterozygotes produced offspring in the expected Mendelian ratios. KO and normal littermates were studied through 2 years of age by hanging latency, rotarod, inked footprint analysis, and histological methods. Disruption of Lix1 expression did not affect survival nor result in any neuromuscular phenotype. Reverse transcriptase-PCR amplification of spinal cord RNA identified a Lix1 alternative transcript beginning in intron 4 and containing exons 5 and 6. The alternative transcript appeared to be rodent specific, and its expression was not disrupted in Lix1 KO mice. Expression of the alternative transcript may have compensated for the loss of Lix1 in the KO mice and thus protected against motor neuron degeneration.