A marked paucity of granule cells in the developing cerebellum of the Npc1(-/-) mouse is corrected by a single injection of hydroxypropyl-ß-cyclodextrin.

In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1(-/-) mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%-25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8-PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1(-/-) mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1(-/-) mice. A single injection of hydroxypropyl-ß-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum.

Frequency comb generation in superconducting resonators.

We have generated frequency combs spanning 0.5 to 20 GHz in superconducting λ/2 resonators at T=3 K. Thin films of niobium-titanium nitride enabled this development due to their low loss, high nonlinearity, low frequency dispersion, and high critical temperature. The combs nucleate as sidebands around multiples of the pump frequency. Selection rules for the allowed frequency emission are calculated using perturbation theory, and the measured spectrum is shown to agree with the theory. Sideband spacing is measured to be accurate to 1 part in 10(8). The sidebands coalesce into a continuous comb structure observed to cover at least several frequency octaves.

Next generation sequencing is here now.

The availability of massively parallel DNA sequencers has brought the cost of sequencing genes to affordable levels but the cost of analyzing the huge amount of data has not decreased to the same extent. Thus, only analyzing the sequences of the genes relevant to the patient's condition makes the cost manageable. A panel of genes relevant to lymphedematous conditions is described.

A novel FLT4 mutation identified in a patient with Milroy disease.

Milroy disease is an autosomal dominant disorder generally presenting with below the knee lymphedema at birth. It is linked to mutations in the tyrosine kinase domain of the VEGFR3 protein which is encoded in the FLT4 gene. Here we report a case of Milroy disease in a patient with a dominant pattern of inheritance, classical physical findings, and lymphatic system imaging demonstrating lack of tracer transport in the lower limbs. Genetic analysis revealed a novel missense mutation compared to a summary of reported mutations causing Milroy Disease.

X-linked CHARGE-like Abruzzo-Erickson syndrome and classic cleft palate with ankyloglossia result from TBX22 splicing mutations.

X-linked cleft palate (CPX) is caused by mutations in the gene encoding the TBX22 transcription factor and is known to exhibit phenotypic variability, usually involving either a complete, partial or submucous cleft palate, with or without ankyloglossia. This study hypothesized a possible involvement of TBX22 in a family with X-linked, CHARGE-like Abruzzo-Erickson syndrome, of unknown etiology. The phenotype extends to additional features including sensorineural deafness and coloboma, which are suggested by the Tbx22 developmental expression pattern but not previously associated in CPX patients. A novel TBX22 splice acceptor mutation (c.593-5T>A) was identified that tracked with the phenotype in this family. A novel splice donor variant (c.767+5G>A) and a known canonical splice donor mutation (c.767+1G>A) affecting the same exon were identified in patients with classic CPX phenotypes and were comparatively analyzed using both in silico and in vitro splicing studies. All three variants were predicted to abolish normal mRNA splicing and an in vitro assay indicated that use of alternative splice sites was a likely outcome. Collectively, the data showed the functional effect of several novel intronic splice site variants but most importantly confirms that TBX22 is the gene underlying Abruzzo-Erickson syndrome, expanding the phenotypic spectrum of TBX22 mutations.

Cholesterol metabolism-associated molecules in late onset Alzheimer's disease.

Alzheimer’s disease (AD) is the most common cause of dementia and, with an aging population, poses a huge public health problem. Although a small per cent is caused by single gene changes, most AD is sporadic and unexplained. Of many modifying factors, changes in brain cholesterol homeostasis are the best studied. We present a review of the role of altered cholesterol metabolism and hypercholesterolemia in APP processing and Abeta generation. We also provide an overview of the potential pharmacological modulation of cholesterol homeostasis in the brain by cholesterol-lowering agents and beta-cyclodextrins.

The Re-Discovery of Dural (Meningeal) Lymphatics: Amnesia or Ambition?

In 2015 the discovery of meningeal (dural) lymphatics was announced to much fanfare. The journal Science named this the second most important discovery of the year! Yet, they had actually been well described two and a quarter centuries earlier, in Italy, England, and Holland. However, there was controversy about their existence because of the difficulties in studying them, also addressed two and a quarter centuries earlier. Their study had generated a very large literature and they were "textbook" knowledge. The reasons for this neglect are discussed emphasizing the current scientific milieu and the changing modes of evaluating scientists.

LYMPHSPIRATION: The importance of the study of dermatoglyphics for lymphologists.

A recent genome-wide association study (GWAS) looking for the genes determining fingerprint and palmar crease patterns disclosed one gene, among many others, which causes lymphedema (CELSR1) while others influencing tissue growth. Since digital fluid influences the height of the volar pads, influences of lymphedema on dermatoglyphics should be sought.

Centrifugal Versus Centripetal Origin(s) of the Lymphatic System: Controversy (Mostly) Resolved?

New findings reopen the controversy about centrifugal vs. centripetal origin of the lymphatic system and support that the latter may be the predominant source of lymphatic endothelial cells from mesenchymal lymphangioblasts.

Human chromosome map of lymphedema-lymphangiogenesis genes: Template for current and future discovery.

We have created a human chromosomal map of the location of known and candidate genes involved in primary lymphedema (PLE). This should facilitate further discovery and provide a basis for understanding microdeletions which cause lymphedema.

Abnormal lymphatic phenotype in a CRISPR mouse model of the human lymphedema-causing Connexin47 R260C point mutation.

Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p<0.01). Associated lymphatic channels were increased in Cx47R259C mice and mesenteric lymph reflux occurred in homozygous Cx47R259C mice but not in wildtype. Contractility of superficial cervical lymphatics, assessed by pressure myography, was reduced in homozygous Cx47R259C mice compared to wildtype. In conclusion, our data are the first to demonstrate a role for the Cx47 protein in lymphatic anatomy and function. This phenotype is similar to that found with other valve deficient mouse mutants, e.g., in Foxc2. Of significance, this study is the first to use CRISPR technology to develop a pre-clinical model of primary lymphedema and demonstrates the importance of distinguishing between lack of and presence of mutant protein when developing clinically relevant animal models for translation of pre-clinical findings.

Genomic duplication resulting in increased copy number of genes encoding the sister chromatid cohesion complex conveys clinical consequences distinct from Cornelia de Lange.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a multisystem congenital anomaly disorder. Heterozygous point mutations in three genes (NIPBL, SMC3 and SMC1A), encoding components of the sister chromatid cohesion apparatus, are responsible for approximately 50-60% of CdLS cases. Recent studies have revealed a high degree of genomic rearrangements (for example, deletions and duplications) in the human genome, which result in gene copy number variations (CNVs). CNVs have been associated with a wide range of both Mendelian and complex traits including disease phenotypes such as Charcot-Marie-Tooth type 1A, Pelizaeus-Merzbacher, Parkinson, Alzheimer, autism and schizophrenia. Increased versus decreased copy number of the same gene can potentially cause either similar or different clinical features. METHODS AND RESULTS: This study identified duplications on chromosomes 5 or X using genome wide array comparative genomic hybridisation (aCGH). The duplicated regions contain either the NIPBL or the SMC1A genes. Junction sequences analyses revealed the involvement of three genomic rearrangement mechanisms. The patients share some common features including mental retardation, developmental delay, sleep abnormalities, and craniofacial and limb defects. The systems affected are the same as in CdLS, but clinical manifestations are distinct from CdLS; particularly the absence of the CdLS facial gestalt. CONCLUSIONS: The results confirm the notion that duplication CNV of genes can be a common mechanism for human genetic diseases. Defining the clinical consequences for a specific gene dosage alteration represents a new "reverse genomics" trend in medical genetics that is reciprocal to the traditional approach of delineation of the common clinical phenotype preceding the discovery of the genetic aetiology.

Lactate dehydrogenase X: effects of antibody on mouse gametes but not on early development.

A rabbit antiserum specific for LDH-X, the spermatozoal form of mouse lactate dehydrogenase, was prepared. This antiserum had no effect on fertility of female mice when injected before or after insemination. Similarly, there was no toxicity to the embryo when high concentrations of the antiserum were added to cultures of 2-cell and 8- to 16-cell embryos. There was, however, a moderate inhibitory effect on fertilization in vitro, which may be attributable to a direct action of antiserum to LDH-X on sperm.

Uniparental disomy and the phenotype of mosaic trisomy 20: a new case and review of the literature.

Mosaic trisomy 20 is one of the most commonly reported chromosome abnormalities detected prenatally, but is rare postnatally. Many studies have hypothesized that uniparental disomy (UPD) may play a role in phenotype variability, but this has not been widely studied. Here we report an additional case of mosaic trisomy 20 with altered pigmentation, in which UPD was not found, and we review the literature.

Expression of Npc1 in glial cells corrects sterility in Npc1(-/-) mice.

Niemann-Pick type C1 (NPC) disease is an autosomal recessive neurodegenerative disorder. One feature of the mouse model of NPC1 is it's infertility. We have made transgenic mice which express the Npc1 protein exclusively in fibrillary astrocytes, using the glial fibrillary acidic protein (GFAP) promoter. This selective expression of Npc1 corrects sterility in GFAP-Npc1(-/-), Npc1(-/-) mice. Counts of acidophils in the pituitary of GFAP-Npc1E, Npc1(-/-) mice, as compared Npc1(-/-) mice, and measurements of dopamine D2 receptor (DRD2) mRNA in the pituitary, suggest mechanisms for fertility enhancement. We conclude that the correction of sterility in GFAP-Npc1E, Npc1(-/-) mice is a result of restoring hypothalamic control of the pituitary.

Lymphedema-distichiasis syndrome without FOXC2 mutation: evidence for chromosome 16 duplication upstream of FOXC2.

A patient with the classical phenotype of Lymphedema-Distichiasis syndrome (OMIM 153400) is described who showed no mutations in the sequence of FOXC2. Accordingly, a Gene Chip 250k array analysis was undertaken with dense SNP genotyping of the genomic region surrounding the FOXC2 locus on Chromosome 16 followed by copy number evaluation by real time PCR. The latter assay showed evidence of a duplicated region 5' of FOXC2 that could be causative for the patient's striking phenotype, which included both distichiasis and a hyperplastic refluxing lymphatic vascular and lymph node phenotype associated with pubertal onset lymphedema, scoliosis and strabismus.

Novel FOXC2 missense mutation identified in patient with lymphedema-distichiasis syndrome and review.

Lymphedema-distichiasis (OMIM 153400) is a dominantly inherited disorder typically presenting with lymphedema at puberty and distichiasis at birth. The condition has been decisively linked to mutations in the forkhead transcription factor FOXC2 which have been primarily frameshift mutations truncating the protein. We report here a novel missense mutation along with a literature review summarizing reported mutations.

From childhood onset lymphedema to fatal fetal hydrops: Possible modifying genes for a FOXC2 mutation.

We performed whole exome sequencing in a family with FOXC2 mutation where the phenotype in one generation was strikingly more severe. Although there were 3 mutations shared by 2 fatal fetal hydrops cases and not the mildly affected mother, none of them were likely to be the cause of the marked phenotypic change.

Post-meiotic gene expression.

Evolutionary arguments and well-designed experiments (based on false premises, however) had suggested that post-meiotic gene expression did not occur in animals. The techniques of molecular genetics have now clearly demonstrated such genetic activity in mammalian testes. The current problem is to understand why some classes of genes, such as Zfy and many oncogenes, are expressed in this manner.

Correction of renal tubular acidosis in carbonic anhydrase II-deficient mice with gene therapy.

Carbonic anhydrase II (CAII) deficiency in humans is associated with a syndrome of renal tubular acidosis, osteopetrosis, and cerebral calcification. A strain of mice of CAII deficiency due to a point mutation also manifests renal tubular acidosis. We report here that retrograde injection of cationic liposome complexed with a CAII chimeric gene, using a cytomegalovirus (CMV) promoter/enhancer as an expression cassette to drive human CAII cDNA, into the renal pelvis of CAII-deficient mice results in expression of CAII in the kidney. The levels of both the CAII gene and its corresponding mRNA were highest by day 3 after treatment, diminishing thereafter, but remaining detectable by 1 mo. After gene therapy, CAII-deficient mice restored the ability to acidify urine after oral administration of ammonium chloride. The ability to acidify urine was maintained at 3 wk after gene therapy, and was eventually lost by 6 wk. Immunohistochemistry studies using anti-CAII antibodies showed that CAII was expressed in tubular cells of the outer medulla and corticomedullary junction. The gene therapy was not associated with nephrotoxicity as assessed by blood urea nitrogen levels and renal histology. To our knowledge, this is the first successful gene therapy of a genetic renal disease. Our results demonstrate the potential of gene therapy as a novel treatment for hereditary renal tubular defects.