Prospective multimodal imaging assessment of locally advanced cervical cancer patients administered by chemoradiation followed by radical surgery-the "PRICE" study 2: role of conventional and DW-MRI.

OBJECTIVES: To assess the diagnostic performance of conventional and DW-MRI parameters in the detection of residual tumor in locally advanced cervical cancer (LACC) patients treated with neoadjuvant chemoradiotherapy (nCRT) and radical surgery METHODS: Between October 2010 and June 2014, 88 patients with histologically documented cervical cancer (FIGO stage IB2-IVA) were prospectively included in the study. Maximum tumor diameters (maxTD), tumor volume (TV), DWI signal intensity (SI), and ADCmean were evaluated at MRI after nCRT. Histology was the reference standard. Treatment response was classified as complete (CR) or partial (PR). Comparisons were made with Mann-Whitney, χ2, and Fisher's exact tests. ROC curves were generated for variables to evaluate diagnostic ability to predict PR and to determine the best cutoff value to predict PR. For each diagnostic test, sensitivity, specificity, and accuracy were calculated. RESULTS: TV and maxTD were significantly smaller in the CR than in the PR group (p < 0.001; p = 0.001) and showed, respectively, sensitivity of 68.8%, specificity of 72.5%, and accuracy of 70.5% and of 47.9, 87.5, and 65.9% in predicting PR. High DWI SI was more frequent in the PR (81.8%) than in the CR group (55.3%) (p < 0.009). ADCmean was higher in the CR (1.3 × 10-3 mm2/s, range 0.8-1.6 × 10-3 mm2/s) than in the PR group (1.1 × 10-3 mm2/s; range 0.7-1.8 × 10-3 mm2/s) (p < 0.018). High DWI SI showed sensitivity, specificity, and accuracy of 81.8, 44.7, and 64.6% in predicting PR. The ADCmean measurement increased sensitivity, specificity, and accuracy to 75.0, 76.2, and 75.4%. CONCLUSIONS: Conventional and DW-MRI is useful for predicting PR after nCRT in LACC. The ADCmean value ≤ 1.1 × 10-3 mm2/s was the best cutoff to predict PR. KEY POINTS: • Conventional and DW-MRI is useful for predicting PR after nCRT in LACC. • The combination of T2 sequences, DW-MRI, and the quantitative measurement of ADC mean showed the best results in predicting pathological PR. • The best cutoff for predicting pathological PR was ADCmeanvalue ≤ 1.1 × 10-3 mm2/s.

17q12 microduplications: a challenge for clinicians.

In the recent years, some cases of 17q12 deletions and duplications have been reported, but the clinical impact of these imbalances is still to be fully elucidated. In particular, 17q12 duplications elude syndrome classification, since they are associated with a wide phenotypic spectrum, ranging from very mild to quite severe phenotypes. Here, two unrelated patients with the same 1.2 Mb microduplication of 17q12 are reported. Comparing these patients' phenotype with those previously published, it emerges that the more patients reported, the more difficult is finding common characteristics, even in presence of exactly the same genetic anomaly. The role of the genes duplicated in this region and the impact of this chromosomal imbalance are discussed.

The QKI-PLP pathway controls SIRT2 abundance in CNS myelin.

Sirtuin 2 (SIRT2), a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase expressed by oligodendrocytes (OLs), the myelin-producing cells of the central nervous system (CNS), is markedly up-regulated during active myelination (Li et al. (2007) J Neurosci 27:2606-2616; Southwood et al. (2007) Neurochem Res 32:187-195; Werner et al. (2007) J Neurosci 27:7717-7730). SIRT2 is a component of the myelin proteome and is severely reduced in the Plp1 knockout mouse brain, in which both proteolipid protein (PLP) and DM20 are absent (Werner et al. (2007) J Neurosci 27:7717-7730). The mechanisms that regulate SIRT2 expression in OLs and myelin remain to be investigated. We report for the first time that the expression of SIRT2 is regulated by the QKI-dependent pathway and this effect is mediated through selective regulation of PLP. In the homozygous quakingviable (qk(v) /qk(v) ) mutant mouse that harbors QKI deficiency in OLs (Bockbrader and Feng (2008) Future Neurol 3:655-668; Ebersole et al. (1996) Nat Genet 12:260-265; Hardy et al. (1996) J Neurosci 16:7941-7949), PLP, but not DM20 mRNA, was selectively down-regulated and SIRT2 protein was severely reduced whereas SIRT2 mRNA expression was unaffected. Expression of the cytoplasmic isoform QKI6 in OLs (Zhao et al. (2006) J Neurosci 26:11278-11286) rescued SIRT2 expression in the qk(v) /qk(v) mutant concomitantly with restoration of PLP expression. Moreover, SIRT2 protein is diminished in myelin tracts and compact myelin of the PLP-ISEdel mutant brain, in which PLP protein but not DM20 is selectively reduced (Wang et al. (2008) Exp Neurol 214:322-330). In contrast, SIRT2 expression and its cellular function in regulating process complexity are not affected by the absence of PLP in PLP-ISEdel non-myelinating OLs. Collectively, our results indicate that the abundance of SIRT2 in myelin is dependent on PLP, but not DM20.

Haplotypes and gene expression implicate the MAPT region for Parkinson disease: the GenePD Study.

BACKGROUND: Microtubule-associated protein tau (MAPT) has been associated with several neurodegenerative disorders including forms of parkinsonism and Parkinson disease (PD). We evaluated the association of the MAPT region with PD in a large cohort of familial PD cases recruited by the GenePD Study. In addition, postmortem brain samples from patients with PD and neurologically normal controls were used to evaluate whether the expression of the 3-repeat and 4-repeat isoforms of MAPT, and neighboring genes Saitohin (STH) and KIAA1267, are altered in PD cerebellum. METHODS: Twenty-one single-nucleotide polymorphisms (SNPs) in the region of MAPT on chromosome 17q21 were genotyped in the GenePD Study. Single SNPs and haplotypes, including the H1 haplotype, were evaluated for association to PD. Relative quantification of gene expression was performed using real-time RT-PCR. RESULTS: After adjusting for multiple comparisons, SNP rs1800547 was significantly associated with PD affection. While the H1 haplotype was associated with a significantly increased risk for PD, a novel H1 subhaplotype was identified that predicted a greater increased risk for PD. The expression of 4-repeat MAPT, STH, and KIAA1267 was significantly increased in PD brains relative to controls. No difference in expression was observed for 3-repeat MAPT. CONCLUSIONS: This study supports a role for MAPT in the pathogenesis of familial and idiopathic Parkinson disease (PD). Interestingly, the results of the gene expression studies suggest that other genes in the vicinity of MAPT, specifically STH and KIAA1267, may also have a role in PD and suggest complex effects for the genes in this region on PD risk.

Herbicide exposure modifies GSTP1 haplotype association to Parkinson onset age: the GenePD Study.

BACKGROUND: Polymorphisms in the glutathione S-transferase pi gene (GSTP1), encoding GSTP1-1, a detoxification enzyme, may increase the risk of Parkinson disease (PD) with exposure to pesticides. Using the GenePD Study sample of familial PD cases, we explored whether GSTP1 polymorphisms were associated with the age at onset of PD symptoms and whether that relation was modified by exposure to herbicides. METHODS: Seven single-nucleotide polymorphisms (SNPs) were genotyped and tested for association with PD onset age in men in three strata: no exposure to herbicides, residential exposure to herbicides, and occupational exposure to herbicides. Haplotypes were similarly evaluated in stratified analyses. RESULTS: Three SNPs were associated with PD onset age in the group of men occupationally exposed to herbicides. Three additional SNPs had significant trends for the association of PD onset age across the herbicide exposure groups. Haplotype results also provided evidence that the relation between GSTP1 and onset age is modified by herbicide exposure. One haplotype was associated with an approximately 8-years-earlier onset in the occupationally exposed group and a 2.8-years-later onset in the nonexposed group. CONCLUSIONS: Herbicide exposure may be an effect modifier of the relation between glutathione S-transferase pi gene polymorphisms and onset age in familial PD.

Skin biopsies demonstrate MPZ splicing abnormalities in Charcot-Marie-Tooth neuropathy 1B.

OBJECTIVE: To demonstrate that intronic mutations in the myelin protein zero (MPZ) cause Charcot-Marie-Tooth neuropathy 1B (CMT1B) by disrupting MPZ splicing. METHODS: We report a family with a T>G transversion at the invariant + 2 position in intron 4 of MPZ (c.614 + 2T>G) that abolishes 5' donor site recognition and is predicted to alter MPZ splicing. We obtained detailed clinical and neurophysiologic analysis of the family. We performed skin biopsies to investigate splicing abnormalities, MPZ protein levels, and localization in myelinated nerves. RESULTS: Patients developed a late onset neuropathy with minimally slow nerve conduction velocities. Skin biopsies confirmed the predicted skipping of exon 4 and downstream frameshift of the mutant MPZ. Quantitative immuno-EM demonstrated normal nerve MPZ levels, suggesting that the mutant MPZ was transported to compact myelin. CONCLUSIONS: Intronic mutations cause CMT1B by disrupting splicing and certain MPZ mutations may cause neuropathy by interacting with the wild type MPZ in the extracellular space of compact myelin.

Further evidence for a fourth gene causing X-linked pure spastic paraplegia.

X-linked hereditary spastic paraplegias (HSPs) present with two distinct phenotypes: pure and complicated. The pure form is characterized by slowly progressive weakness and spasticity of the lower limbs, whereas the complicated forms have additional features (optic neuropathy, retinopathy, extrapyramidal disturbance, dementia, epilepsy, ataxia, ichthyosis, mental retardation, and deafness). Three X-linked loci have been identified for the complicated HSP, while mutations in the proteolipid gene (PLP) (locus SPG2) were implicated in both pure and complicated forms. The absence of identified mutations in the PLP gene in families with both complicated and pure HSP, linked to the SPG2 locus, suggests the existence of another gene in close proximity. We had previously reported a large pedigree with an X-linked form of pure HSP affecting 24 males [Zatz et al., 1976: J Med Genet 13:217-222]. Here, we present the results of linkage analysis in 19 members of this Brazilian family with markers in or near the PLP locus. Positive LOD scores were obtained with markers at the PLP locus (Zmax = 2.41 at Theta = 0); however, no mutation was found in the coding region of PLP, the intron-exon boundaries, or part of the promoter region. The possibility of a duplication of the PLP gene was also excluded. These results suggest either that there is another X-linked gene in close proximity to the PLP gene or that a novel mutation in the noncoding regions of the PLP gene may cause the disease in this family.

Differentiation of glial cells and motor neurons during the formation of neuromuscular junctions in cocultures of rat spinal cord explant and human muscle.

Motor axons extending from embryonic rat spinal cord explants form fully mature neuromuscular junctions with cocultured human muscle. This degree of maturation is not observed in muscle innervated by dissociated motor neurons. Glial cells present in the spinal cord explants seem to be, besides remaining interneurons, the major difference between the two culture systems. In light of this observation and the well documented role of glia in neuronal development, it can be hypothesized that differentiated and long-lived neuromuscular junctions form in vitro only if their formation is accompanied by codifferentiation of neuronal and glial cells and if this codifferentiation follows the spatial and temporal pattern observed in vivo. Investigation of this hypothesis necessitates the characterization of neuronal and glial cell development in spinal cord explant-muscle cocultures. No such study has been reported, although these cocultures have been used in numerous studies of neuromuscular junction formation. The aim of this work was therefore to investigate the temporal relationship between neuromuscular junction formation and the differentiation of neuronal and glial cells during the first 3 weeks of coculture, when formation and development of the neuromuscular junction occurs in vitro. The expression of stage-specific markers of neuronal and glial differentiation in these cocultures was characterized by immunocytochemical and biochemical analyses. Differentiation of astrocytes, Schwann cells, and oligodendrocytes proceeded in concert with the differentiation of motor neurons and neuromuscular junction formation. The temporal coincidence between maturation of the neuromuscular junction and lineage progression of neurons and glial cells was similar to that observed in vivo. These findings support the hypothesis that glial cells are a major contributor to maturity of the neuromuscular junction formed in vitro in spinal cord explant-muscle cocultures.

Novel mutations in spastin gene and absence of correlation with age at onset of symptoms.

Autosomal dominant hereditary spastic paraplegia is genetically heterogeneous, with at least five loci identified by linkage analysis. Recently, mutations in spastin were identified in SPG4, the most common locus for dominant hereditary spastic paraplegia that was previously mapped to chromosome 2p22. We identified five novel mutations in the spastin gene in five families with SPG4 mutations from North America and Tunisia and showed the absence of correlation between the predicted mutant spastin protein and age at onset of symptoms.

Gtx, an oligodendrocyte-specific homeodomain protein, has repressor activity.

Myelin, a multilamellar membrane structure that facilitates nerve conduction, is synthesized in the central nervous system (CNS) by oligodendrocytes. Gtx, a member of the homeodomain family of transcriptional factors, is a candidate regulator of myelin gene expression, because it is uniquely expressed in myelinating oligodendrocytes in postnatal rodent brain. To analyze the regulatory activity of Gtx, we first identified the optimal Gtx-binding sequence using an in vitro DNA-binding assay. This sequence, (A/T)TTAATGA, contains a TAAT core and is similar, but not identical, to that of other homeodomain protein binding sites. When coexpressed in cultured cells along with a minimal promoter containing five tandem repeats of this optimal Gtx-binding sequence, Gtx demonstrated repressor activity, which was also present when Gtx was tethered to DNA by way of the strong GAL4 DNA-binding domain. Truncations of the GAL4-Gtx fusion identified a portable repressor domain within a relatively proline/alanine-rich region N-terminal to the Gtx homeodomain. Cotransfection of a Gtx expression vector into a variety of cell lines, including oligodendrocytes, along with constructs containing portions of the PLP, MBP, or Gtx promoters fused to a reporter gene, however, did not modulate transcription from any of these promoter constructs. These data support the notion that the oligodendrocyte-specific homeodomain protein Gtx can act as a transcriptional repressor. In addition, they suggest that interaction of Gtx with other, as yet undefined, transcriptional regulators modifies Gtx activity in oligodendrocytes.

Peripheral neuropathy caused by proteolipid protein gene mutations.

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system typically caused by duplications or missense mutations of the proteolipid protein (PLP) gene. Most investigators have found that peripheral nerve function and structure is normal in PMD patients. We have found that null mutations of the PLP gene cause demyelinating peripheral neuropathy, whereas duplications and a proline 14 to leucine mutation do not affect nerve function. A family with a nonsense mutation at position 144, which affects only PLP but not the alternatively spliced gene product DM20, has a very mild syndrome, including normal peripheral nerve function. Our findings suggest that DM20 alone is sufficient to maintain normal nerve function and that there may be domains of PLP/DM20 that have a relatively more active role in the peripheral nervous system compared with that in the central nervous system.

The molecular pathogenesis of Pelizaeus-Merzbacher disease.

In 1885, Pelizaeus described 5 boys in a single family with nystagmus, spastic quadriparesis, ataxia, and delay in cognitive development. In 1910, Merzbacher reexamined this family, which then included 14 affected individuals, including 2 girls, and found that all affected family members shared a common female ancestor. Also, he noted that the disease was passed exclusively through the female line without male-to-male transmission. Pathological analysis of brain tissue from one affected individual showed that most of the central white matter lacked histochemical staining for myelin, although there were occasional small regions of preserved myelin, giving the sections a "tigroid" appearance. The description of this family provides the clinical, genetic, and pathological basis for Pelizaeus-Merzbacher disease (PMD): an X-linked disorder of myelination classically characterized by nystagmus, spastic quadriparesis, ataxia, and cognitive delay in early childhood.

Cell cycle arrest induced by ectopic expression of p27 is not sufficient to promote oligodendrocyte differentiation.

Oligodendrocyte differentiation is accompanied by dramatic changes in gene expression as well as cell cycle arrest. To determine whether cell cycle arrest is sufficient to induce the changes in cell phenotype associated with differentiation, we inhibited oligodendrocyte precursor proliferation in vitro by overexpressing p27, a cyclin kinase inhibitor, using a recombinant adenovirus. Ectopic expression of p27 efficiently inhibited oligodendrocyte precursor cell division, even in the presence of exogenous mitogens, by blocking the activity of the cyclin-dependent kinase, cdk2. Although the cells had stopped dividing, they did not express galactocerebroside (GalC) or myelin basic protein (MBP), changes associated with oligodendrocyte differentiation, suggesting that they had not differentiated. After removal of exogenous mitogens, however, adenovirus-expressing oligodendrocyte precursors differentiated with a temporal profile similar to that of control, uninfected oligodendrocytes, as indicated by expression of GalC and MBP. We conclude that cell cycle arrest is not sufficient to induce differentiation of dividing oligodendrocyte precursors, and that modulation of additional, as yet unknown, signaling pathways is required for this to occur.

Changes in the activity of cdk2 and cdk5 accompany differentiation of rat primary oligodendrocytes.

Oligodendrocytes, the myelinating cells of the central nervous system, are terminally differentiated cells that originate through asynchronous waves of proliferation and differentiation of precursors present at birth. Withdrawal from cell cycle and onset of differentiation are tightly linked and depend on an intrinsic program modulated by the action of growth factors. p27 plays a central and obligatory role in the initiation of oligodendrocyte differentiation and cessation of proliferation. In this paper, we have characterized the role of modulation of cdk2 and cdk5 kinase activity during the process of oligodendrocyte precursor differentiation. As rat primary oligodendrocytes differentiate in culture there is a fall in cdk2 activity and a rise in cdk5 activity as well as an increase in the cdk inhibitor, p27 protein. The decline in cdk2 activity is not accompanied by a drop in cdk2 protein level, suggesting that it results from inhibition of cdk2 activation rather than decreased protein expression. Taken together, these data suggest that oligodendrocytes may withdraw from the cell cycle at G1-S transition through inactivation of cdk2 activity, possibly initiated by increasing amount of p27, and that cdk5 may have a role until now unrecognized in the differentiation of oligodendrocytes.

Proteolipid protein is necessary in peripheral as well as central myelin.

Alternative products of the proteolipid protein gene (PLP), proteolipid protein (PLP) and DM20, are major components of compact myelin in the central nervous system, but quantitatively minor constituents of Schwann cells. A family with a null allele of PLP has a less severe CNS phenotype than those with other types of PLP mutations. Moreover, individuals with PLP null mutations have a demyelinating peripheral neuropathy, not seen with other PLP mutations of humans or animals. Direct analysis of normal peripheral nerve demonstrates that PLP is localized to compact myelin. This and the clinical and pathologic observations of the PLP null phenotype indicate that PLP/DM20 is necessary for proper myelin function both in the central and peripheral nervous systems.

Successful pregnancy in a neurologically impaired woman with Wilson's disease.

Patients with treated Wilson's disease and no residual impairments should have successful pregnancies. We report a case of a neurologically impaired patient with Wilson's disease who had a successful pregnancy with no complications. This is the first case to document that compliance with penicillamine therapy as low as 500 mg/day avoids placental and fetal copper accumulation.

Hereditary spastic paraplegia: advances in genetic research. Hereditary Spastic Paraplegia Working group.

Hereditary spastic paraplegia (HSP) is a diverse group of inherited disorders characterized by progressive lower-extremity spasticity and weakness. Insight into the genetic basis of these disorders is expanding rapidly. Uncomplicated autosomal dominant, autosomal recessive, and X-linked HSP are genetically heterogeneous: different genes cause clinically indistinguishable disorders. A locus for autosomal recessive HSP is on chromosome 8q. Loci for autosomal dominant HSP have been identified on chromosomes 2p, 14q, and 15q. One locus (Xq22) has been identified for X-linked, uncomplicated HSP and shown to be due to a proteolipoprotein gene mutation in one family. The existence of HSP families for whom these loci are excluded indicates the existence of additional, as yet unidentified HSP loci. There is marked clinical similarity among HSP families linked to each of these loci, suggesting that gene products from HSP loci may participate in a common biochemical cascade, which, if disturbed, results in axonal degeneration that is maximal at the ends of the longest CNS axons. Identifying the single gene defects that cause HSPs distal axonopathy may provide insight into factors responsible for development and maintenance of axonal integrity. We review clinical, genetic, and pathologic features of HSP and present differential diagnosis and diagnostic criteria of this important group of disorders. We discuss polymorphic microsatellite markers useful for genetic linkage analysis and genetic counseling in HSP.

Refined genetic mapping and proteolipid protein mutation analysis in X-linked pure hereditary spastic paraplegia.

X-linked hereditary spastic paraplegias (HSP) present with two distinct phenotypes, pure and complicated. The pure form is characterized by spasticity and gait difficulties but lacks the additional features (nystagmus, dysarthria, mental retardation) present in the complicated form. The complicated form is heterogeneous, caused by mutations of the L1CAM gene at Xq28 (SPG1) or the PLP gene at Xq22 (SPG2) that is allelic to Pelizaeus-Merzbacher disease (PMD). Since in one kindred (K313) the pure form of HSP was also mapped to Xq22, this raises the issue as to whether a pure form of HSP exists that is allelic to X-linked complicated HSP (SPG2) and PMD. To answer this question, we carried out linkage analysis in a new pedigree with pure HSP (K101) and refined linkage in pedigree K313. The PLP gene was also screened for mutation by direct sequencing and reverse-transcriptase polymerase chain reaction (RT-PCR). In both families, the disease locus mapped to Xq22 with Lod scores at zero recombination of 5.3 for COL4A5 2B6 in K313 and 2.4 for DXS101 in K101. A T to C transition in exon 5 of the PLP gene was identified from affected individuals of K313. This transition causes a Ser to Pro mutation in the major extracellular loop of PLP/DM20. This finding demonstrates that a form of X-linked pure spastic paraplegia, X-linked complicated HSP (SPG2) and PMD are allelic disorders. There was no evidence of mutations in either coding sequences or the intron/exon junctions of PLP in pedigree K101, suggesting that the disease-producing mutation may be in the noncoding portions of PLP or in a nearby gene.

X-linked pure familial spastic paraparesis. Characterization of a large kindred with magnetic resonance imaging studies.

OBJECTIVE: Families with pure X-linked familial spastic paraparesis are rare. We describe a large kindred with the "pure" form of X-linked familial spastic paraparesis with seven clinically affected males. The current study was designed to identify the presence of nuclear magnetic resonance imaging (MRI) abnormalities in the affected individuals. PATIENTS AND METHODS: Twenty-three individuals were examined, and MRIs of the brain were obtained in all seven affected males and two females. RESULTS: The disease is characterized by spastic gait and increased reflexes without other associated neurologic signs. No male-to-male transmission has been documented in this pedigree. Magnetic resonance images of the brain in affected individuals demonstrate discrete white matter lesions in the periatrial regions, more prominent posteriorly. Similar, although not as extensive, white matter lesions were detected in the brain of the single obligate female carrier studied with MRI. CONCLUSIONS: We report previously undescribed (to our knowledge) findings of MRI in pure X-linked familial spastic paraparesis and discuss the use of MRI in the diagnosis of this disorder and as a possible screening study of potential carriers.