Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features

Background Congenital deletions affecting 3q11q23 have rarely been reported and only five cases have been molecularly characterised. Genotype. phenotype correlation has been hampered by the variable sizes and breakpoints of the deletions. In this study, 14 novel patients with deletions in 3q11q23 were investigated and compared with 13 previously reported patients. Methods Clinical data were collected from 14 novel patients that had been investigated by high resolution microarray techniques. Molecular investigation and updated clinical information of one cytogenetically previously reported patient were also included. Results The molecular investigation identified deletions in the region 3q12.3q21.3 with different boundaries and variable sizes. The smallest studied deletion was 580 kb, located in 3q13.31. Genotype. phenotype comparison in 24 patients sharing this shortest region of overlapping deletion revealed several common major characteristics including significant developmental delay, muscular hypotonia, a high arched palate, and recognisable facial features including a short philtrum and protruding lips. Abnormal genitalia were found in the majority of males, several having micropenis. Finally, a postnatal growth pattern above the mean was apparent. The 580 kb deleted region includes five RefSeq genes and two of them are strong candidate genes for the developmental delay: DRD3 and ZBTB20. Conclusion A newly recognised 3q13.31 microdeletion syndrome is delineated which is of diagnostic and prognostic value. Furthermore, two genes are suggested to be responsible for the main phenotype.

Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion

Background Split-hand/foot malformation (SHFM)-also known as ectrodactyly-is a congenital disorder characterised by severe malformations of the distal limbs affecting the central rays of hands and/or feet. A distinct entity termed SHFLD presents with SHFM and long bone deficiency. Mouse models suggest that a defect of the central apical ectodermal ridge leads to the phenotype. Although six different loci/mutations (SHFM1-6) have been associated with SHFM, the underlying cause in a large number of cases is still unresolved. Methods High resolution array comparative genomic hybridisation (CGH) was performed in patients with SHFLD to detect copy number changes. Candidate genes were further evaluated for expression and function during limb development by whole mount in situ hybridisation and morpholino knock-down experiments. Results Array CGH showed microduplications on chromosome 17p13.3, a locus previously associated with SHFLD. Detailed analysis of 17 families revealed that this copy number variation serves as a susceptibility factor for a highly variable phenotype with reduced penetrance, particularly in females. Compared to other known causes for SHFLD 17p duplications appear to be the most frequent cause of SHFLD. A similar to 11.8 kb minimal critical region was identified encompassing a single gene, BHLHA9, a putative basic loop helix transcription factor. Whole mount in situ hybridisation showed expression restricted to the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knock down of bhlha9 in zebrafish resulted in shortening of the pectoral fins. Conclusions Genomic duplications encompassing BHLHA9 are associated with SHFLD and non-Mendelian inheritance characterised by a high degree of non-penetrance with sex bias. Knock-down of bhlha9 in zebrafish causes severe reduction defects of the pectoral fin, indicating a role for this gene in limb development.