Consolato Sergi

Mutations in WDR62, encoding a centrosome-associated protein, cause microcephaly with simplified gyri and abnormal cortical architecture

Genes associated with human microcephaly, a condition characterized by a small brain, include critical regulators of proliferation, cell fate and DNA repair. We describe a syndrome of congenital microcephaly and diverse defects in cerebral cortical architecture. Genome-wide linkage analysis in two families identified a 7.5-Mb locus on chromosome 19q13.12 containing 148 genes. Targeted high throughput sequence analysis of linked genes in each family yielded > 4,000 DNA variants and implicated a single gene, WDR62, as harboring potentially deleterious changes. We subsequently identified additional WDR62 mutations in four other families. Magnetic resonance imaging and postmortem brain analysis supports important roles for WDR62 in the proliferation and migration of neuronal precursors. WDR62 is a WD40 repeat–containing protein expressed in neuronal precursors as well as in postmitotic neurons in the developing brain and localizes to the spindle poles of dividing cells. The diverse phenotypes of WDR62 suggest it has central roles in many aspects of cerebral cortical development.

Study of the malformation of ductal plate of the liver in Meckel syndrome and review of other syndromes presenting with this anomaly.

Meckel syndrome (MIM 249.000) is an autosomal recessive disorder with a variable spectrum of anomalies. Since the first reports of this syndrome, very broad diagnostic criteria have been proposed, but the process of defining them continues. It is probable that at least two of three manifestations, including cystic kidney dysplasia, occipital encephalocele or other anomaly of the central nervous system, and postaxial polydactyly occur in most cases. Arrest of the development of intrahepatic bile ducts at the stage of the bilaminar plate formation or ductal plate malformation is considered of high diagnostic value in Meckel syndrome, but there is no complete agreement in the literature about its occurrence. The aims of this investigation were to study the prevalence and morphologic patterns of ductal plate malformation of the liver in Meckel syndrome by evaluating the dilatation of primitive biliary structures and the increase in connective tissue of the portal tract. Archival data files from four German centers (Berlin, Freiburg, Heidelberg, Mainz) were reviewed. Liver sections of 30 well-studied fetuses with Meckel syndrome were immunostained with antibodies against cytokeratins (intermediate filaments of the cytoskeleton) and factor VIII (an endothelial cell marker) and were evaluated both qualitatively and quantitatively. Cystic kidney dysplasia, occipital encephalocele, and postaxial polydactyly were found in 100%, 90%, and 83.3% of the fetuses, respectively. Ductal plate malformation of the liver was a constant anomaly in Meckel syndrome, seen as frequently as renal lesions. We observed essentially two kinds of hepatic lesions: 23 cases showed mainly a cystic dilatation of primitive biliary structures with little portal fibrosis, while 7 cases showed mainly rings of interrupted curved lumina around a central fibrovascular axis and pronounced portal fibrosis. In these seven cases an abnormal pattern of the portal vein, with many small and closely spaced branches of the portal vein (the so-called pollard willow pattern), was also seen. With respect to other fetal developmental anomalies, no difference between the two types of lesions was found. We also provide a potentially useful comprehensive review of other genetic syndromes in which ductal plate malformations may occur.

β-cell development and turnover during prenatal life in humans

INTRODUCTION beta-cell regeneration is an area under active investigation for the future treatment of diabetes, but little is known about the patterns and dynamics of prenatal beta-cell development in humans. In particular, the quantitative changes in beta-cell mass in the developing pancreas have not been elucidated in detail. We addressed the following questions in prenatal humans: i) what is the timing of beta-cell occurrence and islet growth? ii) What are the dynamics of beta-cell replication and apoptosis? METHODS Pancreatic tissue was obtained from 65 human embryos and foetuses aged between 8 weeks post conception (p.c.) and birth. Sections were stained for insulin, glucagon, Ki67 (proliferation marker), TUNEL (apoptosis marker) and CD31 (blood vessel marker), and morphometric analyses were performed. RESULTS beta-cells were detected from gestational week 9 onward, whereas glucagon expression was detected already at week 8. The fractional beta-cell area of the pancreas increased in a linear fashion until birth (r=0.60, P<0.001). The first endocrine cells were found within or adjacent to the primitive ductal epithelium. beta-cell replication was readily detected in the newly forming islets already starting at week 9 p.c. (average frequency 2.8+/-0.4%). A small percentage of cells co-expressed insulin and glucagon during the early foetal period. There was a close relationship between the development of endocrine islets and blood vessels during all stages of prenatal pancreas development suggesting a possible interaction between both cell types. The frequency of beta-cell apoptosis was relatively high throughout all ages (1.5+/-0.3%). CONCLUSIONS beta-cell differentiation in humans occurs from week 9 p.c. onward. The first endocrine cells are closely associated with the ductal epithelium suggesting differentiation from precursor cells. High rates of beta-cell replication suggest that this mechanism plays an important role in the prenatal expansion of beta-cell mass.

Polymorphisms of the BRAF gene predispose males to malignant melanoma

The incidence of malignant melanoma has rapidly increased in recent years. Evidence points to the role of inheritance in melanoma development, but specific genetic risk factors are not well understood. Recent reports indicate a high prevalence of somatic mutations of the BRAF gene in melanomas and melanocytic nevi. Here we report that germ-line single nucleotide polymorphisms (SNPs) in BRAF are significantly associated with melanoma in German males, but not females. At-risk haplotypes of BRAF are shown. Based upon their frequencies, we estimate that BRAF could account for a proportion attributable risk of developing melanoma of 4% in the German population. The causal variant has yet to be determined. The burden of disease associated with this variant is greater than that associated with the major melanoma susceptibility locus CDKN2A, which has an estimated attributable risk of less than 1%.

OTX2 mutations contribute to the otocephaly-dysgnathia complex

Background Otocephaly or dysgnathia complex is characterised by mandibular hypoplasia/agenesis, ear anomalies, microstomia, and microglossia; the molecular basis of this developmental defect is largely unknown in humans. Methods and results This study reports a large family in which two cousins with micro/anophthalmia each gave birth to at least one child with otocephaly, suggesting a genetic relationship between anophthalmia and otocephaly. OTX2, a known microphthalmia locus, was screened in this family and a frameshifting mutation was found. The study subsequently identified in one unrelated otocephalic patient a sporadic OTX2 mutation. Because OTX2 mutations may not be sufficient to cause otocephaly, the study assayed the potential of otx2 to modify craniofacial phenotypes in the context of known otocephaly gene suppression in vivo. It was found that otx2 can interact genetically with pgap1, prrx1, and msx1 to exacerbate mandibular and midline defects during zebrafish development. However, sequencing of these loci in the OTX2-positive families did not unearth likely pathogenic lesions, suggesting further genetic heterogeneity and complexity. Conclusion Identification of OTX2 involvement in otocephaly/dysgnathia in humans, even if loss of function mutations at this locus does not sufficiently explain the complex anatomical defects of these patients, suggests the requirement for a second genetic hit. Consistent with this notion, trans suppression of otx2 and other developmentally related genes recapitulate aspects of the otocephaly phenotype in zebrafish. This study highlights the combined utility of genetics and functional approaches to dissect both the regulatory pathways that govern craniofacial development and the genetics of this disease group.

Bone morphogenetic protein-2 and growth and differentiation factor-5 enhance the healing of necrotic bone in a sheep model.

Abstract Introduction: Osteotropic growth factors enhance bone repair, but their efficacy in an area of necrotic bone is not known. The purpose of this study was to investigate the effects and potential side effects of an intraosseous application of absorbable bone morphogenetic protein-2 (BMP-2) and growth and differentiation factor-5 (GDF-5) composites in a sheep model for partial necrosis of the femoral head. Materials and methods: The direct injection of ethanol under fluoroscopy into the superior centre of the right femoral head produced histologically documentable necrosis of the central region of the head in a previous study of ten sheep. Another 27 sheep constituted the sample to study the effects of BMP-2 and GDFJ. Necrosis was produced in the same fashion in these animals. Four weeks later nine sheep received 300 μg recombinant BMP-2 and nine sheep 300 μg recombinant GDF-5 on an absorbable carrier by surgical implantation. Nine sheep received the carrier alone (control group). The animals were sacrificed at 3, 6, and 12 weeks after implantation and both femora were harvested. Results: Bone density analysis and microscopic examination indicated that bone formation was noticeably induced as early as 3 weeks postoperatively in the growth factor treated animals. Bone regeneration was enhanced by growth factor composites. This was documented by histological scoring and histomorphometric analysis. No severe local side effects secondary to the growth factors, such as heterotopic ossification or inflammation, were observed in either group. Discussion: The application of an absorbable growth factor composite in combination with established surgical techniques is a promising approach, that may enhance the healing of devitalised bone defects. Based on these results, further studies regarding biodegradation, dosage of the protein and surgical technique are required.

A new animal model of femoral head necrosis induced by intraosseous injection of ethanol

There is no reliable animal model of the early stages of osteonecrosis of the femoral head (ONFH) for the evaluation of new therapeutic approaches. In this study, we propose a new animal model of femoral head osteonecrosis. Pure ethanol was injected into the centre of the femoral head in adult Merino sheep under fluoroscopic control. After 3, 6 and 12 weeks the animals were killed and the femoral heads were harvested. Microradiographic and histological changes were analysed and recorded. Partial necrosis was documented over a period of 12 weeks in all animals. The appearance of necrosis in combination with intact macrotexture, macrocirculation and joint cartilage is similar to the features described in early ONFH in humans. Due to its efficacy and its similarity to the early stages of ONFH in humans, this model may be suitable to evaluate new therapeutic techniques in the treatment of ONFH.

The remodeling of the primitive human biliary system

From 12 weeks of gestation on, a progressive remodeling of the human primitive biliary structure or ductal plate occurs. A few parts of the primitive biliary structure (peripheral tubular or ductular structures) dilate, migrate toward the center of the portal tract, and transform into mature bile ducts, while most of them gradually disappear. To the best of our knowledge, quantitative studies have been performed only to evaluate the ratio between the number of remodeled bile ducts and portal tracts during human fetal development. We studied the remodeling of the intrahepatic fetal biliary structures as well as the bile duct to portal tract ratio in the developing human liver by immunohistochemistry with monoclonal antibodies anti-bile duct type cytokeratins and using a computer-based image-analysis system. We found that the surface and the perimeter of the portal tracts, the longest axis of the migrating peripheral tubular structures, and the maturation of bile ducts follow a process continuous and active up to term, but they slow between the 20th and the 32nd week of gestation, when intraportal granulopoiesis of the liver is active.

ARLTS1 variants and melanoma risk

Variants in the tumor suppressor gene ARLTS1 (ADP‐ribosylation factor‐like tumor‐suppressor gene 1) have been shown to influence familial cancer risk. Both Cys148Arg and Trp149Stop were associated with an increased risk of familial or high‐risk familial breast cancer, respectively. We studied the impact of these gene variants on melanoma risk, investigating 351 melanoma patients and 804 control subjects. While ARLTS1 Trp149Stop did not influence melanoma risk (OR = 0.83, 95% CI = 0.37–1.88, p = 0.65), Cys148Arg revealed a statistically significant association with an increased risk for heterozygous carriers (OR = 1.43, 95% CI = 1.05–1.95, p = 0.02). An additional risk enhancement, though statistically non‐significant, was observed in individuals with multiple melanomas (OR = 2.33, 95% CI = 0.87–6.26, p = 0.08).

Huge fetal sacrococcygeal teratoma with a completely formed eye and intratumoral DNA ploidy heterogeneity.

ABSTRACT Preterm infants and newborns with sacrococcygeal teratomas are at high risk for prenatal and perinatal complications. The prognosis depends on size and histology of the tumor, degree of prematurity, associated malformations, route of delivery, and prompt and complete surgical removal. Virtually any tissue can be present in a sacrococcygeal teratoma, but to date, ocular lens has been documented only as lentinoids (lens-like cells), whereas flow-cytometric data have been variably interpreted. We describe a case of a sacrococcygeal teratoma in an infant of 29 weeks gestational age that is remarkable for the weight (4500 g), the presence of a completely formed eye, and intratumoral DNA ploidy heterogeneity.