Generation and characterization of two human iPSC lines from patients with methylmalonic acidemia cblB type.

Two human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of two siblings with methylmalonic acidemia cblB type carrying mutations in the MMAB gene: c.287T➔C (p.Ile96Thr) and a splicing loss-of-function variant c.584G➔A affecting the last nucleotide of exon 7 in MMAB (p.Ser174Cysfs*23). Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability.

Adjuvant treatment for pancreatic ductal carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with a very poor prognosis. Most of the patients are diagnosed in advanced stages of the disease, and 5-year survival rates in these patients remains <10%. Surgery still remains the only radical treatment option, although only 15-20% of patients are candidates for surgical resection at the time of the diagnosis. Patients who undergo radical surgery still have a limited survival rate, being the average of 23 months. Three clinical trials have shown that adjuvant chemotherapy therapy after surgery may improve survival: CONKO-1, ESPAC-3, and ESPAC-4. Adjuvant therapy is recommended in patients with R0/R1, T1-4/N1-0 tumors and with ECOG 0-1. In patients with ECOG-2, the decision needs to be individualized. Treatment schemes that have demonstrated efficacy include gemcitabine alone, 5-fluorouracil, or the combination of gemcitabine and capecitabine for six months. Prior to adjuvant treatment, the following test are recommended: Complete blood tests, including CA19.9 biomarker; imaging studies to rule out early disease relapse (preferable thorax-abdomen-pelvic CT). Studies that have evaluated the efficacy of radiation therapy in the adjuvant setting have presented conflicting results. Its use should be considered in patients with R1 or R2 tumors or in those with lymph nodes involved.

Consensus guidelines for diagnosis, treatment and follow-up of patients with pancreatic cancer in Spain.

The management of patients with pancreatic cancer has advanced over the last few years. We convey a multidisciplinary group of experts in an attempt to stablish practical guidelines for the diagnoses, staging and management of these patients. This paper summarizes the main conclusions of the working group. Patients with suspected pancreatic ductal adenocarcinoma should be rapidly evaluated and referred to high-volume centers. Multidisciplinary supervision is critical for proper diagnoses, staging and to frame a treatment plan. Surgical resection together with chemotherapy offers the highest chance for cure in early stage disease. Patients with advanced disease should be classified in treatment groups to guide systemic treatment. New chemotherapeutic regimens have resulted in improved survival. Symptomatic management is critical in this disease. Enrollment in a clinical trial is, in general, recommended.

Six9 (Optx2), a new member of the six gene family of transcription factors, is expressed at early stages of vertebrate ocular and pituitary development.

The Drosophila gene sine oculis (so) is a nuclear homeoprotein, which is required for eye development. Several homologues of so have been found in vertebrates. We report here a detailed expression analysis in chick and mouse of Six9 (Optx2), a novel gene of the Six/sine oculis family closely related to Six3. Six9 (Optx2) is first expressed at presomitic stages in the head-fold, both in the neural plate and in the underlying axial mesoderm. Thereafter, Six9 (Optx2) is strongly expressed in the presumptive and differentiating neural retina and ventral optic stalk, in the olfactory placodes, in the hypothalamus and in the pituitary gland. This expression pattern largely overlaps with that of Six3, but several differences exist between the expression domain of the two genes. At presomitic stages, the posterior boundary of Six3 expression is at the same axial level both in the prechordal plate and in the overlying neural plate. In contrast, Six9 (Optx2) expression in the prechordal plate extends more caudal to that of the neural plate, occupying a more restricted V-shaped territory. Similarly, during the early events of eye patterning, Six3 is first expressed in the entire optic vesicle and lens placode. Only later does its expression become confined to the prospective and differentiating neural retina. Conversely, Six9 (Optx2) is never observed in the lens placode of either chick and mouse, and from early stages of optic vesicle development, Six9 (Optx2) transcripts are restrained to the prospective ventral neural retina and optic stalks.

Designing recombinant Pseudomonas strains to enhance biodesulfurization.

The dsz biodesulfurization cluster from Rhodococcus erythropolis IGTS8 has been engineered under the control of heterologous broad-host-range regulatory signals to alleviate the mechanism of sulfur repression, and it was stably inserted into the chromosomes of different Pseudomonas strains. The recombinant bacteria were able to desulfurize dibenzothiophene more efficiently than the native host. Furthermore, these new biocatalysts combine relevant industrial and environmental traits, such as production of biosurfactants, with the enhanced biodesulfurization phenotype.

Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome.

Hemolytic-uremic syndrome (HUS) is a microvasculature disorder leading to microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Most cases of HUS are associated with epidemics of diarrhea caused by verocytotoxin-producing bacteria, but atypical cases of HUS not associated with diarrhea (aHUS) also occur. Early studies describing the association of aHUS with deficiencies of factor H suggested a role for this complement regulator in aHUS. Molecular evidence of factor H involvement in aHUS was first provided by Warwicker et al., who demonstrated that aHUS segregated with the chromosome 1q region containing the factor H gene (HF1) and who identified a mutation in HF1 in a case of familial aHUS with normal levels of factor H. We have performed the mutational screening of the HF1 gene in a novel series of 13 Spanish patients with aHUS who present normal complement profiles and whose plasma levels of factor H are, with one exception, within the normal range. These studies have resulted in the identification of five novel HF1 mutations in four of the patients. Allele HF1 Delta exon2, a genomic deletion of exon 2, produces a null HF1 allele and results in plasma levels of factor H that are 50% of normal. T956M, W1183L, L1189R, and V1197A are missense mutations that alter amino acid residues in the C-terminal portion of factor H, within a region--SCR16-SCR20--that is involved in the binding to solid-phase C3b and to negatively charged cellular structures. This remarkable clustering of mutations in HF1 suggests that a specific dysfunction in the protection of cellular surfaces by factor H is a major pathogenic condition underlying aHUS.

Genomic cloning, structure, expression pattern, and chromosomal location of the human SIX3 gene.

The Drosophila gene sine oculis (so) is a nuclear homeoprotein that is required for eye development. Homologous genes to so, denoted SIX genes, have been found in vertebrates. Among the SIX genes, SIX3 is considered to be the functional homologue of so. To provide insight into the potential implications of SIX3 in human ocular malformations, we have cloned and characterized the human SIX3 gene. In human eye, SIX3 produces a 3-kb transcript that codes for a 332-amino-acid polypeptide that is virtually identical to its mouse and chick homologues. Expression of SIX3 was detected in human embryos as early as 5-7 weeks of gestation and found to be maintained in the eye throughout the entire period of fetal development. At 20 weeks of gestation, expression of SIX3 in the human retina was detected in the ganglion cells and in cells of the inner nuclear layer. The human SIX3 gene spans 4.4 kb of genomic DNA and is split in two exons separated by a 1659-bp intron. SIX3 was mapped to human chromosome 2p16-p21, between the genetic markers D2S119 and D2S288. Interestingly, the map position of human SIX3 overlaps the locations of two dominant disorders with ocular phenotypes that have been assigned to this chromosomal region, holoprosencephaly type 2 and Malattia Leventinese.

Genomic cloning and characterization of the human homeobox gene SIX6 reveals a cluster of SIX genes in chromosome 14 and associates SIX6 hemizygosity with bilateral anophthalmia and pituitary anomalies.

The Drosophila gene sine oculis (so), a nuclear homeoprotein that is required for eye development, has several homologues in vertebrates (the SIX gene family). Among them, SIX3 is considered to be the functional orthologue of so because it is strongly expressed in the developing eye. However, embryonic SIX3 expression is not limited to the eye field, and SIX3 has been found to be mutated in some patients with holoprosencephaly type 2 (HPE2), suggesting that SIX3 has wide implications in head development. We report here the cloning and characterization of SIX6, a novel human SIX gene that is the homologue of the chick Six6(Optx2) gene. SIX6 is closely related to SIX3 and is expressed in the developing and adult human retina. Data from chick and mouse suggest that the human SIX6 gene is also expressed in the hypothalamic and the pituitary regions. SIX6 spans 2567 bp of genomic DNA and is split in two exons that are transcribed into a 1393-nucleotide-long mRNA. Chromosomal mapping of SIX6 revealed that it is closely linked to SIX1 and SIX4 in human chromosome 14q22.3-q23, which provides clues about the origin and evolution of the vertebrate SIX family. Recently three independent reports have associated interstitial deletions at 14q22.3-q23 with bilateral anophthalmia and pituitary anomalies. Genomic analyses of one of these cases demonstrated SIX6 hemizygosity, strongly suggesting that SIX6 haploinsufficiency is responsible for these developmental disorders.

The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism.

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.

Familial syndromic esophageal atresia maps to 2p23-p24.

Esophageal atresia (EA) is a common life-threatening congenital anomaly that occurs in 1/3,000 newborns. Little is known of the genetic factors that underlie EA. Oculodigitoesophageoduodenal (ODED) syndrome (also known as "Feingold syndrome") is a rare autosomal dominant disorder with digital abnormalities, microcephaly, short palpebral fissures, mild learning disability, and esophageal/duodenal atresia. We studied four pedigrees, including a three-generation Dutch family with 11 affected members. Linkage analysis was initially aimed at chromosomal regions harboring candidate genes for this disorder. Twelve different genomic regions covering 15 candidate genes (approximately 15% of the genome) were excluded from involvement in the ODED syndrome. A subsequent nondirective mapping approach revealed evidence for linkage between the syndrome and marker D2S390 (maximum LOD score 4.51 at recombination fraction 0). A submicroscopic deletion in a fourth family with ODED provided independent confirmation of this genetic localization and narrowed the critical region to 7.3 cM in the 2p23-p24 region. These results show that haploinsufficiency for a gene or genes in 2p23-p24 is associated with syndromic EA.

A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2).

Progressive myoclonus epilepsy of the Lafora type or Lafora disease (EPM2; McKusick no. 254780) is an autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration and glycogen-like intracellular inclusion bodies (Lafora bodies). A gene for EPM2 previously has been mapped to chromosome 6q23-q25 using linkage analysis and homozygosity mapping. Here we report the positional cloning of the 6q EPM2 gene. A microdeletion within the EPM2 critical region, present inhomozygosis in an affected individual, was found to disrupt a novel gene encoding a putative protein tyrosine phosphatase (PTPase). The gene, denoted EPM2, presents alternative splicing in the 5' and 3' end regions. Mutational analysis revealed that EPM2 patients are homozygous for loss-of-function mutations in EPM2. These findings suggest that Lafora disease results from the mutational inactivation of a PTPase activity that may be important in the control of glycogen metabolism.