Clericuzio-type poikiloderma with neutropenia syndrome in three sibs with mutations in the C16orf57 gene: delineation of the phenotype.

We report on three sibs who have autosomal recessive Clericuzio-type poikiloderma neutropenia (PN) syndrome. Recently, this consanguineous family was reported and shown to be informative in identifying the C16orf57 gene as the causative gene for this syndrome. Here we present the clinical data in detail. PN is a distinct and recognizable entity belonging to the group of poikiloderma syndromes among which Rothmund-Thomson is perhaps the best described and understood. PN is characterized by cutaneous poikiloderma, hyperkeratotic nails, generalized hyperkeratosis on palms and soles, neutropenia, short stature, and recurrent pulmonary infections. In order to delineate the phenotype of this rare genodermatosis, the clinical presentation together with the molecular investigations in our patients are reported and compared to those from the literature.

Search for genomic imbalances in a cohort of 24 Cornelia de Lange patients negative for mutations in the NIPBL and SMC1L1 genes.

Cornelia de Lange syndrome (CdLS) is a rare, multiple congenital anomaly/mental retardation syndrome characterized by varied clinical signs including facial dysmorphism, pre- and post-natal growth defects, small hands and malformations of the upper limbs. Established genetic causes include mutations in the NIPBL (50-60%), SMC1L1 and SMC3 (5%) genes. To detect chromosomal rearrangements pointing to novel positional candidate CdLS genes, we used array-CGH to analyze a subgroup of 24 CdLS patients negative for mutations in the NIPBL and SMC1L1 genes. We identified three carriers of DNA copy number alterations, including a de novo 15q26.2-qter 8-Mb deletion, and two inherited 13q14.2-q14.3 1-Mb deletion and 13q21.32-q21.33 1.5-Mb duplication, not reported among copy number variants. The clinical presentation of all three patients matched the diagnostic criteria for CdLS, and the phenotype of the patient with the 15qter deletion is compared to that of both CdLS and 15qter microdeletion patients.

Identification of novel genomic markers related to progression to glioblastoma through genomic profiling of 25 primary glioma cell lines.

Identification of genetic copy number changes in glial tumors is of importance in the context of improved/refined diagnostic, prognostic procedures and therapeutic decision-making. In order to detect recurrent genomic copy number changes that might play a role in glioma pathogenesis and/or progression, we characterized 25 primary glioma cell lines including 15 non glioblastoma (non GBM) (I-III WHO grade) and 10 GBM (IV WHO grade), by array comparative genomic hybridization, using a DNA microarray comprising approx. 3500 BACs covering the entire genome with a 1 Mb resolution and additional 800 BACs covering chromosome 19 at tiling path resolution. Combined evaluation by single clone and whole chromosome analysis plus 'moving average (MA) approach' enabled us to confirm most of the genetic abnormalities previously identified to be associated with glioma progression, including +1q32, +7, -10, -22q, PTEN and p16 loss, and to disclose new small genomic regions, some correlating with grade malignancy. Grade I-III gliomas exclusively showed losses at 3p26 (53%), 4q13-21 (33%) and 7p15-p21 (26%), whereas only GBMs exhibited 4p16.1 losses (40%). Other recurrent imbalances, such as losses at 4p15, 5q22-q23, 6p23-25, 12p13 and gains at 11p11-q13, were shared by different glioma grades. Three intervals with peak of loss could be further refined for chromosome 10 by our MA approach. Data analysis of full-coverage chromosome 19 highlighted two main regions of copy number gain, never described before in gliomas, at 19p13.11 and 19q13.13-13.2. The well-known 19q13.3 loss of heterozygosity area in gliomas was not frequently affected in our cell lines. Genomic hotspot detection facilitated the identification of small intervals resulting in positional candidate genes such as PRDM2 (1p36.21), LRP1B (2q22.3), ADARB2 (10p15.3), BCCIP (10q26.2) and ING1 (13q34) for losses and ECT2 (3q26.3), MDK, DDB2, IG20 (11p11.2) for gains. These data increase our current knowledge about cryptic genetic changes in gliomas and may facilitate the further identification of novel genetic elements, which may provide us with molecular tools for the improved diagnostics and therapeutic decision-making in these tumors.

Germline mutation in the juxtamembrane domain of the kit gene in a family with gastrointestinal stromal tumors and urticaria pigmentosa.

BACKGROUND: Gain-of-function mutations of the c-kit protooncogene, mainly clustered in the juxtamembrane domain, have been reported in a significant fraction of gastrointestinal (GI) stromal tumors (GISTs) that represent the most common mesenchymal tumor of the GI tract. Two families also have been described with a GIST predisposition syndrome with a germline c-kit mutation affecting either the juxtamembrane domain or the tyrosine kinase domain. Here, the authors report on a family in which the dominantly inherited trait of hyperpigmented spots was inherited from an individual who developed multiple GISTs with diffuse hyperplasia of the myenteric plexus by his son, who was affected with urticaria pigmentosa. METHODS: Screening for the c-kit mutation was performed by means of polymerase chain reaction-based denaturing gradient gel electrophoresis/constant denaturing gel electrophoresis followed by direct sequencing of abnormal conformers. Expression of KIT and CD34 was determined by immunohistochemistry. RESULTS: In peripheral blood DNA samples, both affected family members showed a previously undescribed c-kit mutation in the juxtamembrane domain, resulting in the substitution of alanine for valine(559). Mutation and polymorphic marker analyses on DNA samples from three GISTs and two skin biopsy specimens evidenced the same mutation in the heterozygous condition. Immunohistochemical examination showed coexpression of CD117 (c-kit) and CD34 in all independent GISTs and CD117 positivity in mast cells from the skin lesions. CONCLUSIONS: Comparative analysis of clinical presentation and mutation mapping in the families described to date point to the peculiar association of mast cells, melanocytic dysfunction, and GIST predisposition in carriers of c-kit mutations within the juxtamembrane domain.

RNA hyperediting and alternative splicing of hematopoietic cell phosphatase (PTPN6) gene in acute myeloid leukemia.

The SH2 domain-containing tyrosine phosphatase PTPN6 (SHP-1, PTP1C, HCP) is a 68 kDa cytoplasmic protein primarily expressed in hematopoietic cell development, proliferation and receptor-mediated mitogenic signaling pathways. By means of direct dephosphorylation, it down-regulates a broad spectrum of growth-promoting receptors, including the Kit tyrosine kinase, activated to elicit a prominent cascade of intracellular events by stem cell factor binding. The pivotal contribution of PTPN6 in modulating myeloid cell signaling has been revealed by the finding that shp-1 mutation is responsible for the overexpansion and inappropriate activation of myelomonocytic populations in motheaten (me/me) and motheaten viable (me(v)/me(v)) mice. Association of PTPN6 with c-Kit and negative modulation of the myeloid leukocyte signal transduction pathways prompted us to examine the expression of the protein tyrosine phosphatase PTPN6 gene in CD34(+)/CD117(+) blasts from acute myeloid leukemia patients. We identified and cloned cDNAs representing novel PTPN6 mRNA species, derived from aberrant splicing within the N-SH2 domain leading to retention of intron 3. Sequence analysis of cDNA clones revealed multiple A-->G editing conversions. The editing of PTPN6 mRNA mainly occurred as an A-->G conversion of A(7866), which represents the putative branch site in IVS3 of PTPN6 mRNA. Evidence that editing of A(7866) abrogates splicing has been obtained in vitro by using an edited clone and its backward clone generated by site-directed mutagenesis. The level of the aberrant intron-retaining splice variant, evaluated by semi-quantitative RT-PCR, was lower in CD117(+)-AML bone marrow mononuclear cells at remission than at diagnosis, suggesting the involvement of post-transcriptional PTPN6 processing in leukemogenesis.

Effects of diets with different content in protein and fiber on embryotoxicity induced by experimental diabetes in rats.

Three groups of streptozotocin-diabetic rats were maintained during pregnancy on three hyperproteic diets with different protein contents. These differences were compensated by an equal quantity of fiber (group 1: protein 55.0%, fiber 4.5%; group 2: 45.0%, 14.0%; group 3: 35.0%, 24.0%). Three groups of nondiabetic pregnant rats were fed with the same diets and served as control. The differences of the daily protein intake among the diabetic groups were less pronounced than those expected on the basis of the diet composition, and the embryopathic effects (reduced fetal weight, increased in malformation and resorption rate) were not statistically different among the three groups of diabetic animals. The frequency of congenital malformations was higher than that observed in a previous experiment in diabetic rats maintained on a standard diet, but much lower than that observed in animals fed on a purified, fiber-poor, normoproteic diet. When the caloric intake of the diabetic rats in the different groups was determined it was found to be similar for all of them and also similar to the caloric intake of the rats given a standard nonteratogenic diet (in previous experiments), while the rats maintained on a normoproteic, teratogenic diet increased their caloric intake. These results seem to indicate that the diet composition greatly influences the intake of food and calories of pregnant diabetic rats and this may play a role in modulating the embryopathic effect of diabetes.

Damage to the crystalline lens in infants of diabetic mothers: a pathology so far neglected?

Congenital cataract occurs in 90-95% of diabetic rat fetuses. The pathogenetic mechanism is triggered by fetal hyperglycemia and presents the following steps: (1) a high glucose concentration in the lens; (2) reduction of glucose to sorbitol by aldose reductase; (3) accumulation of sorbitol into the fibers of the lens creating a hyperosmotic effect, leading to (4) an infusion of liquid into the fibers, which (5) become hydropic and degenerate (vacuolization). This series of manifestations might also occur in fetuses of pregnant diabetic mothers. Post birth glycemia diminishes rapidly, and this favorable condition which decreases vacuolization is perhaps the reason why such degeneration has not yet been observed. Since the fibers of the lens are permanent cells, damage in the fetal period might later bring about negative consequences. We hope that someone will study whether this ocular pathology occurs in human infants born to diabetic mothers.

Diet composition modifies embryotoxic effects induced by experimental diabetes in rats.

Despite improvements in prenatal care, the incidence of congenital malformations in diabetic pregnancies is still 3-4 times higher than in normal pregnancies. These defects could be attributed to alterations of intrauterine environment due to disorder of the maternal metabolism. If this were true, the quality of food could play a role in diabetes-induced embryotoxicity. To check this hypothesis, female CD rats were made diabetic by injecting intravenously 50 mg/kg of streptozotocin 2 weeks before mating. From the first day of pregnancy they were divided into three groups and maintained on the following diets: (1) standard diet (Italiana Mangimi); (2) purified high protein diet (protein 55%, carbohydrates 25.5%, fat 7.5%, fiber 4.5%, ash 7.5%); (3) purified normoprotein diet (protein 19%, carbohydrates 62.5%, fat 7.5%, fiber 4%, ash 7%). Nondiabetic pregnant females fed with standard diet served as negative control. No significant differences were observed in blood glucose levels among the groups (range 410-500 mg/dl). The group fed on normoprotein diet showed at term of pregnancy: (1) higher rate of resorptions; (2) lower fetal weight; (3) higher frequency of major malformations than the groups fed standard and hyperproteic diets. Although we are not able at this time to discriminate between a protective effect of a diet with a high protein content and a disruptive effect of a diet containing high quantity of carbohydrates, the results of this trial support the hypothesis of a fuel-mediated teratogenesis in diabetic pregnancy.

Congenital malformations in offspring of diabetic rats: experimental study on the influence of the diet composition and magnesium intake.

In spite of improvements in the treatment of diabetes, the risk of congenital malformations in diabetic pregnancy is three to four times higher than in normal pregnancy. This might be due to the metabolic abnormalities of diabetic pregnancy that also affect mineral metabolism. Since diabetes can lower both maternal and fetal blood Mg levels, and Mg deficiency has been shown to be teratogenic in laboratory animals, we decided to investigate which effects Mg deficiency would have in inducing embryopathy in diabetic animals. Female CD rats were divided into six groups. Groups 1 and 2 were fed a standard diet (Mg content 4,200 ppm), groups 3-6 a purified diet (Mg contents 4,200, 500, 250, or 125 ppm). Groups 2-6 had been made diabetic by an intravenous injection of 50 mg/kg streptozocin 1 week before mating. The rats were killed on day 21 of pregnancy, and the live fetuses were examined for external, skeletal, and visceral malformations. The maternal and fetal blood glucose levels were the same in all diabetic groups. The maternal Mg levels in groups 2 and 3 were the same as in controls, but definitely lower in groups 4-6. Embryotoxicity (embryonic deaths, delayed development, congenital malformations) was higher in the groups fed the purified diet than in group 2, but without a clear relation to the dietary Mg levels. We cannot draw any conclusions about the effects of Mg deficiency in diabetic pregnancy from our results, but they show that the quality of the diet is of major importance in the manifestation of embryotoxicity in diabetes.

Oversized infant of diabetic mother: its cause and prevention.

In this study the birth weights of 431 infants of diabetic mothers of the Milan series have been compared with the birth weights of infants of a control group. The averages and the centile distributions of weights of infants of gestational diabetic mothers (Class A) and of diabetic mothers without vascular complications (Classes B and C) did not differ substantially from those of control newborns (table I, figure 1). This confirms the clinical indication, based on the hyperglycemia-hyperinsulinism theory that fetal macrosomia can be prevented provided maternal metabolism is strictly controlled. In this series insulin was administered at the maximal tolerated dose (MTD), a therapeutic regimen that provides excellent metabolic control of the mother. In multiparae, the birth weights of the infants of the latest pregnancy were drastically lower than the birth weights of the infants in their previous pregnancies (without MTD insulin) (table II). Our results do not confirm the recent hypothesis that pregnant diabetics with strict metabolic control during pregnancy generally give birth to growth retarded infants. The MTD of insulin has also been administered to gestational diabetic mothers, and fetal macrosomia was prevented (table I, figure 1). This confirms the opinion of those who believe that a diet-regimen must be accompanied by insulin administration to correct the slight metabolic abnormality of these patients. As would be expected because of placental insufficiency, infants of patients with vascular complications, including those who have only calcifications of the pelvic vessels (White' Class E), were growth retarded (table I, figure 1). The risk of fetal growth retardation in Class E has not been remarked upon in the literature, since pathology of pelvic vessels is usually disregarded and the patients remain undifferentiated among Classes A-C. The possibility to prevent fetal macrosomia with a strict control of maternal diabetes has been questioned because of the lack of correlation between fetal macrosomia and the degree of maternal hyperglycemia and of fetal hyperinsulinism. We postulate that, if fetal hyperinsulinism causes hypoxia, as it does in experimental animals, the lack of correlation may be due to the fetal hyperinsulinism itself.

Effects of streptozotocin-induced diabetes on fetal development of the rat.

The purpose of this study was to determine whether or not in rats with experimentally induced diabetes there is an increased frequency of congenital malformations; data in the literature are not consistent on this point. Virgin CD females rats were injected with 40-50 mg/kg streptozotocin (Stz) before mating (SIBM group) or on the first day of pregnancy (SI1). Both SIBM and SI1 females were divided into two groups according to their blood glucose levels: severely diabetic (SD, greater than 300 mg%) and mildly diabetic (MD, 120-250 mg%). Food and water consumption by the control and MD groups were the same, but the SD females developed polyphagia, polyuria, and polydypsia, which continued to increase throughout pregnancy, as did the blood glucose levels. All the MD females mated and carried to term. In SD females both frequency of mating and fertility were only slightly lower than in the controls. All the females were killed on the 21st day of pregnancy. Pre- and postimplantation losses were the same for diabetic and control rats, but SIBM-SD females ovulated less than other groups. Weights of fetuses of SD dams were lower and blood sugar levels higher than those of the other groups. The placentas of SD rats were significantly heavier and there was cystic degeneration of spongiosa. The incidence of major malformations was minimal (approximately 2%) in fetuses of SD females and there were none at all in controls or MD females. In conclusion, our data are in agreement with those of other investigators who have found that rats with experimentally induced diabetes have smaller fetuses and increased placental weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous preterm delivery in pregnant diabetics: a high risk hitherto "unrecognized".

Preterm delivery (PD)--before the 259th day from the beginning of the last menstrual period--is very frequent in pregnant diabetics (from 50% to 80% or more, personal investigation in progress). In these patients a policy is generally followed of a systematically controlled early delivery. Therefore, one is inclined to think that the high frequency of PD is mainly the consequence of this policy. However, it has been recently pointed out [5] that spontaneous labor accounts for half or more of PD in pregnant diabetics. Moreover in pregnant women with gestational diabetes PD rate due to spontaneous labor is three times higher than in the general obstetric population [5]. A different result emerges from our case study, 1963-1975. Insulin was administered to the maximal tolerated dose both in case of gestational and clinical diabetes [1, 6, 7, 8, 9, 10, 11, 12]. The incidence of PD due to spontaneous labor is 6.7% (7.1% in gestational and 6.1% in clinical diabetes), i.e. no difference from PD rate in general. Since 1974 M. Chartier, Paris, has been adopting the same therapeutic criteria [2]. His results seem to confirm that the risk of PD due to spontaneous labor drastically reduces in pregnant diabetics strictly controlled.