Romiplostim for Treatment of Children and Young Adults With Severe Aplastic Anemia and Myelodysplastic Syndrome.

Thrombopoietin receptor agonists (TPO-RAs) induce trilineage hematopoiesis under conditions with acquired hematopoietic failure. We evaluated safety, tolerability, and preliminary efficacy of a TPO-RA, romiplostim (Nplate), with or without standard-of-care immunosuppressive therapy (±IST) for children (ages < 21 y) with newly diagnosed and relapsed/refractory severe aplastic anemia (SAA) and myelodysplastic syndrome (MDS). Data were collected from an observational study and a single arm interventional pilot study. The safety outcome was treatment-related adverse events (AEs). Efficacy was evaluated by complete hematopoietic response (CHR) at week 24. Romiplostim was commenced at 5 µg/kg/week, with dose escalation of 2.5 µg/kg/week (maximum, 20 µg/kg/dose) based on platelet response. Romiplostim was continued until CHR was observed. Ten subjects (SAA, 9 [IST, 4; without IST, 5]; MDS, 1) completed the study (median age: 9.2 y). Median romiplostim dose was 10 µg/kg/week (range: 5 to 17.5 µg/kg/week). The cumulative incidence of CHR was 70.4% (95% CI, 20.2%-92.6%). Among 21 AEs (Grade 1 to 3), 3 were attributed to romiplostim. At a median posttherapy follow-up of 10.9 months (range: 0.7 to 77.5), no clonal evolution, bone marrow fibrosis or mortality was reported. This proof-of-concept study provides data about short-term safety, tolerability, and preliminary efficacy of romiplostim (±IST) for treatment of pediatric SAA/MDS.

Congenital Neutropenia with Specific Granulocyte Deficiency Caused by Novel Double Heterozygous SMARCD2 Mutations.

SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) is critical for myelopoiesis. Recently, bi-allelic SMARCD2 mutations have been reported in five children, causing autosomal recessive congenital neutropenia with specific granulocytes deficiency (CN-SGD); a syndrome resulting in G-CSF resistant neutropenia, recurrent infections, and dysplastic myelopoiesis. We report a new case with CN-SGD caused by two novel heterozygous pathogenic variants in the SMARCD2 gene (c.1081del (p.Gln361Argfs*15)), and (c.217C>T (p.Arg73*)). Treatment with the weekly dosing of thrombopoietin receptor agonist, Romiplostim, along with daily G-CSF transformed her clinical course, implying potential synergism. This report advances the understanding of CN-SGD caused by SMARCD2 mutations.

Complexities of genetic diagnosis illustrated by an atypical case of congenital hypoplastic anemia.

Diamond-Blackfan Anemia (DBA) is a rare polygenic disorder defined by congenital hypoplastic anemia with marked decrease or absence of bone marrow erythroid precursors. Identifying the specific genetic etiology is important for counseling and clinical management. A 6-yr-old boy with a clinical diagnosis of DBA has been followed by our pediatric hematology team since birth. His clinical course includes transfusion-dependent hypoplastic anemia and progressive autoimmune cytopenias. Genetic testing failed to identify a causative mutation in any of the classical DBA-associated genes. He and his parents underwent trio whole-exome sequencing (WES) with no genetic etiology identified initially. Clinical persistence and suspicion led to testing for adenosine deaminase 2 (ADA2) activity and whole-genome sequencing (WGS) that identified compound heterozygous pathogenic mutations in the ADA2-encoding CECR1 gene, a recently appreciated etiology for congenital hypoplastic anemia. This case illustrates current challenges in genetic testing and how they can be overcome by multidisciplinary expertise in clinical medicine and genomics.

ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells.

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study, we demonstrate that Y-27632, a specific inhibitor of Rho kinase (ROCK) activity, significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore, treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably, hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y-27632. Additionally, we determined that Y-27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus, Y-27632 provides a means to "kick-start" slow-growing human pluripotent stem cells, especially after being thawed from frozen stocks. Together, these results argue that Y-27632 is a useful tool in overcoming obstacles to studies involving the cultivation of both hES cells and human iPS cells.

Regulation of the Nanog gene by both positive and negative cis-regulatory elements in embryonal carcinoma cells and embryonic stem cells.

The transcription factor Nanog is essential for mammalian embryogenesis, as well as the pluripotency of embryonic stem (ES) cells. Work with ES cells and embryonal carcinoma (EC) cells previously identified positive and negative cis-regulatory elements that influence the activity of the Nanog promoter, including adjacent cis-regulatory elements that bind Sox2 and Oct-3/4. Given the importance of Nanog during mammalian development, we examined the cis-regulatory elements required for Nanog promoter activity more closely. In this study, we demonstrate that two positive cis-regulatory elements previously shown to be active in F9 EC cells are also active in ES cells. We also identify a novel negative regulatory region that is located in close proximity to two other positive Nanog cis-regulatory elements. Although this negative regulatory region is active in F9 EC cells and ES cells, it is inactive in P19 EC cells. Furthermore, we demonstrate that one of the positive cis-regulatory elements active in F9 EC cells and ES cells is inactive in P19 EC cells. Together, these and other studies suggest that Nanog transcription is regulated by the interplay of positive and negative cis-regulatory elements. Given that P19 appears to be more closely related to a later developmental stage of mammalian development than F9 and ES cells, differential utilization of cis-regulatory elements may reflect mechanisms used during development to achieve the correct level of Nanog expression as embryogenesis unfolds.

Expansions of CAG.CTG repeats in immortalized human astrocytes.

Expansions of trinucleotide repeats (TNRs) are the genetic cause for a number of neurodegenerative disorders. In some of these diseases, ongoing somatic expansions in the brain are thought to contribute to disease progression. Expansions can occur in both neurons and supporting glial cells, but little is known about molecular mechanisms of expansion in these cells, particularly glia. To help address this issue, a cultured human astrocyte cell line called SVG-A was tested for expansions of CAG*CTG repeats present on a shuttle vector. A quantitative genetic selection showed that +4 to +15 repeat expansions occur readily for starting alleles of 25 repeats, thereby spanning the important boundary between short stable repeats and longer more unstable CAG*CTG tracts. These expansions in glial cell culture, as in humans, were sequence and length-dependent, and were inhibited by the presence of a sequence interruption within the triplet repeat tract. These findings suggest that the mutations seen in cell culture reflect at least some of the in vivo expansions seen in glia. Mechanistically, it was found that the direction of DNA replication through the TNR influenced the frequency of expansions, suggesting that either replication or a replication-associated process, such as DNA repair, contributes to CAG*CTG tract instability in SVG-A cells. This finding is consistent with the idea that replication-based mechanisms can be a source of TNR expansions in astrocytes, which, unlike neurons, retain proliferative capacity throughout life.

Cellular, molecular and clinical characterization of patients with Hermansky-Pudlak syndrome type 5.

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelles such as melanosomes and platelet dense granules. Seven genes are now associated with HPS in humans. An accurate diagnosis of each HPS subtype has important prognostic and treatment implications. Here we describe the cellular, molecular, and clinical aspects of the recently identified HPS-5 subtype. We first analyzed the genomic organization and the RNA expression pattern of HPS5, located on chromosome 11p14, and demonstrated tissue-specific expression of at least three alternatively spliced HPS5 mRNA transcripts, coding for HPS5A and HPS5B proteins, that differ at their 5'-ends. Genetic screening of 15 unassigned HPS patients yielded six new HPS5 mutations in four patients. Clinically, our HPS-5 patients exhibited iris transillumination, variable hair and skin pigmentation, and absent platelet dense bodies, but not pulmonary fibrosis or granulomatous colitis. In two patients with homozygous missense mutations, hemizygosity was ruled out by gene-dosage multiplex polymerase chain reaction, and immunocytochemical analyses of their fibroblasts supported the HPS-5 diagnosis. Specifically, LAMP-3 distribution was restricted to the perinuclear region in HPS-5 fibroblasts, in contrast to the normal LAMP-3 distribution, which extended to the periphery. This specific intracellular vesicle distribution in fibroblasts, in combination with the clinical features, will improve the characterization of the HPS-5 subtype.

Hermansky-Pudlak syndrome type 4 (HPS-4): clinical and molecular characteristics.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder of oculocutaneous albinism and bleeding attributable to storage-pool-deficient platelets. Although at least 14 mouse models of HPS exist, the human disorders that comprise HPS, i.e., HPS-1, HPS-2, HPS-3, and HPS-4, are recognized to result from mutations in four genes, viz., HPS1, ADTB3A, HPS3, and HPS4, respectively. To characterize further the recently identified HPS-4 disease on molecular and clinical grounds, we first identified the genomic organization of HPS4, located on chromosome 22q11.2-q12.2, including its intron/exon boundaries. We found that HPS4 produces at least two alternatively spliced mRNA transcripts that differ at their 5'-ends. Next, we performed an extensive analysis of 22 unassigned HPS patients (i.e., not having HPS-1, HPS-2, or HPS-3 disease). Using single-strand conformation polymorphism, we determined that seven of the 22 patients had HPS-4. In these seven individuals, we identified five different HPS4 mutations, including one frameshift insertion, one missense, and three nonsense mutations. Three alleles in two patients contained the previously reported Q698insAAGCA frameshift. Three HPS4 mutations were newly described. Four alleles in three patients contained R217X, and two siblings were compound heterozygotes for E138X and E222X. Clinically, our HPS-4 patients exhibited iris transillumination, variable hair and skin pigmentation, absent platelet dense bodies, and occasional pulmonary fibrosis and granulomatous colitis, a severe phenotype similar to that of patients with HPS-1.