Defects in long-range regulatory elements have recently emerged as previously underestimated factors in the genesis of human congenital disorders. Léri-Weill dyschondrosteosis is a dominant skeletal malformation syndrome caused by mutations in the short stature homeobox gene SHOX. We have analysed four families with Léri-Weill dyschondrosteosis with deletions in the pseudoautosomal region but still with an intact SHOX coding region. Using fluorescence in situ hybridization and single nucleotide polymorphism studies, we identified an interval of approximately 200 kb that was deleted in all tested affected family members but retained in the unaffected members and in 100 control individuals. Comparative genomic analysis of this interval revealed eight highly conserved non-genic elements between 48 and 215 kb downstream of the SHOX gene. As mice do not have a Shox gene, we analysed the enhancer potential in chicken embryos using a green fluorescent protein reporter construct driven by the beta-globin promoter, by in ovo electroporation of the limb bud. We observed cis-regulatory activity in three of the eight non-genic elements in the developing limbs arguing for an extensive control region of this gene. These findings are consistent with the idea that the deleted region in the affected families contains several distinct elements that regulate Shox expression in the developing limb. Furthermore, the deletion of these elements in humans generates a phenotype apparently undistinguishable to those patients identified with mutations in the SHOX coding region and, for the first time, demonstrates the potential of an in vivo assay in chicken to monitor putative enhancer activity in relation to human disease.

SummaryAn observational population-based cohort study was performed to investigate the role of comorbidity on outcome and treatment-related toxicity in patients with newly diagnosed advanced-stage diffuse large B-cell lymphoma (DLBCL) treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). Data for the clinical characteristics of 154 patients (median age 69 years), including Charlson Comorbidity Index (CCI), treatment, toxicity and outcome were evaluated. Forty-five percent of the patients had an International Prognistic index ≥3 and 16% had a CCI ≥2. The planned R-CHOP schedule was completed by 84% and 75% reached complete remission (CR). In those with CCI ≥2, 67% completed treatment with 46% CR. In patients with a CCI <2, overall survival (OS) after 1, 2 and 5 years was 84%, 79% and 65% respectively and it was 64%, 48% and 48% for those with CCI ≥2. Grade III/IV toxicity was documented in 53%, most frequently febrile neutropenia (27%) and infections (23%). In multivariate analysis CCI ≥2 and IPI ≥3 were independent risk indicators for OS and grade III/IV toxicity. In conclusion, comorbidity is an independent risk indicator for worse OS in patients with advanced DLBCL treated with R-CHOP by interference with intensive treatment schedules and more grade III/IV toxicity. Future studies are warranted to determine the optimal treatment approach in patients with significant comorbidities.

To determine the clonal nature of hematopoiesis and to assess lineage involvement in patients with myelodysplastic syndromes (MDS), we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Eleven female MDS patients heterozygous for at least one of these probes were studied: 3 with refractory anemia (RA), 2 with RA with ringed sideroblasts (RARS), 2 with chronic myelomonocytic leukemia (CMML), and 4 with RA with excess of blasts in transformation (RAEB-t). All exhibited clonal hematopoiesis as determined by Southern analysis of DNA prepared from peripheral blood (PB) and/or bone marrow (BM) cells. In three of the six patients heterozygous for the PGK1 gene, purified cell suspensions of polymorphonuclear cells (PMN), monocytes, lymphocytes, and/or T cells prepared from PB were tested. In addition, five of these patients were analyzed by a polymerase chain reaction (PCR)-based procedure as described recently. This method was slightly adapted to facilitate the analysis of cell lysates of fluorescence- activated cell sorted (FACS) monocytes, T and B lymphocytes, and natural killer (NK) cells. The outcome of Southern and PCR analysis was concordant, showing that PMN and monocytes were clonally derived, whereas circulating T and B lymphocytes and NK cells exhibited random X- chromosome inactivation compatible with a polyclonal pattern. To address the question of whether T cells are derived from unaffected progenitor cells or that their origin had antedated the onset of MDS, naive and memory T cells were analyzed separately. Both subsets showed a polyclonal pattern. However, in one patient analysis of constitutive DNA suggested a skewed methylation, and the presence of clonal lymphocytes against a background of polyclonal lymphoid cells cannot be ruled out in this patient. PCR analysis of PB and BM cells showed a nonrandom, unilateral pattern of X-inactivation, compatible with a mixture of clonally (myeloid) and polyclonally (lymphoid) derived cells. In conclusion, in some patients, MDS represents a disorder with clonal hematopoiesis restricted to cells of myeloid origin, whereas a random X-inactivation pattern is found in lymphoid cells.

Background Although congenital adrenal hyperplasia (CAH) is known to be associated with adrenal crises (AC), its association with patient or clinician reported sick day episodes (SDE) is less clear. Methods Data on children with classic 21-hydroxylase deficiency CAH from 34 centers in 18 countries, of which 7 were Low or Middle Income (LMIC) and 11 were High Income (HIC), were collected from the International CAH Registry and analysed to examine the clinical factors associated with SDE and AC. Results 518 children with a median of 11 children (range 1, 53) per center had 5,388 visits evaluated over a total of 2,300 patient years. The median number of AC and SDE per patient year per center was 0 (0, 3) and 0.4 (0.0, 13.3), respectively. Of the 1,544 SDE, an AC was reported in 62 (4%), with no fatalities. Infectious illness was the most frequent precipitating event, reported in 1,105 (72%) and 29 (47%) of SDE and AC, respectively. On comparing cases from LMIC and HIC, the median SDE per patient year was 0.75 (0, 13.3) vs 0.11 (0, 12.0) (p&lt;0.001), respectively and the median AC per patient year was 0 (0, 2.2) vs 0 (0, 3.0) (p=0.43), respectively. Conclusions The real-world data that are collected within the I-CAH Registry show wide variability in the reported occurrence of adrenal insufficiency related adverse events. As these data become increasingly used as a clinical benchmark in CAH care, there is a need for further research to improve and standardise the definition of SDE.

Clonality of myeloid and lymphoid cell fractions obtained from peripheral blood (PB) or bone marrow (BM) of five patients with a myelodysplastic syndrome (MDS), was studied by combined immunophenotypic analysis and DNA in situ hybridization. This novel technique enables quantitative and direct analysis of cytogenetic alterations in nondividing cells of distinct cell lineages. Four patients with a trisomy 8 and one patient with a translocation (1;7) were studied. For cell lineage determination, antibodies specific for progenitor cells (CD34), myeloid cells (CD15), monocytes (63D3), T cells (CD3), and B cells (CD19,20,22) were used. In one patient with a trisomy 8, BM cells were available and the erythroid lineage could be studied. For detection of cytogenetic aberrations, we used chromosome- specific repetitive DNA probes. In three patients, all nonlymphoid cells carried the cytogenetic abnormality; in two patients, mosaicism within these lineages was suggested by the relative low numbers (35% to 55%) of aberrant cells. None of the T or B cells of the five patients carried the chromosomal aberrations. We conclude that combined immunophenotyping and in situ hybridization is a feasible technique to study lineage involvement. Our data suggest that the chromosomal aberrations studied in MDS are restricted to the myeloid lineages.