Mohammed Faruq

Prognostic value of MIB-1, p53, epidermal growth factor receptor, and INI1 in childhood chordomas

BACKGROUND Chordomas are slow-growing tumors and most commonly involve the sacrum and clivus. Multiple recurrences are frequent. Childhood chordomas are rare and often show exceptionally aggressive behavior, resulting in short survival and a high incidence of metastatic spread. OBJECTIVE This study examined the histologic features and immunohistochemical profile of pediatric chordomas and compared them with their adult counterparts. METHODS Nine pediatric and 13 adult cases were included in the study. Childhood chordomas were classified into conventional, atypical, and poorly differentiated types. Immunohistochemistry was performed for cytokeratin, epithelial membrane antigen, vimentin, S100, brachyury, p53, INI1, epidermal growth factor receptor (EGFR), and CD117. Cytogenetic analyses were performed in a subset of tumors for SMARCB1/INI1 locus on 22q chromosome by fluorescent in situ hybridization (FISH) and analysis of the SMARCB1/INI1 gene sequence. RESULTS All tumors showed expression of cytokeratin, epithelial membrane antigen, S100, vimentin, brachyury, and EGFR. Atypical morphology, p53 expression, higher MIB-1 labelling index (LI), and INI1 loss were more frequently seen in pediatric chordomas as compared with adults. None of the tumors showed CD117 expression. No point mutation in the SMARCB1/INI1 gene was noted in the tumors examined; however, 4 pediatric and 1 adult chordoma showed loss of this locus on FISH analysis. CONCLUSIONS A subset of pediatric chordomas with atypical histomorphologic features needs to be identified, as they behave in an aggressive manner and require adjuvant therapy. Pediatric chordomas more frequently show p53 expression, INI1 loss, and higher MIB-1 LI as compared with adults, whereas EGFR expression is common to both.

Gray matter volume deficits in spinocerebellar ataxia: An optimized voxel based morphometric study ☆

INTRODUCTION Spinocerebellar ataxias (SCA) are a group of autosomal dominant ataxias with varied clinical phenotypes. However there are no unique distinguishing features on routine neuroimaging among the various genetically defined SCAs. Voxel-based morphometry (VBM) provides an automated unbiased analysis of structural MRI scans and gives a comprehensive assessment of anatomical differences throughout the brain. OBJECTIVES The aims of this study were to (i) characterize the patterns of atrophy in SCA1, SCA2 and SCA3 using optimized VBM, (ii) demonstrate the characteristic anatomical differences in these genetically distinct SCA subtypes, and (iii) assess the relationship between morphometric measures and the CAG repeat lengths and other attributes of the disease. METHODS Thirty-one genetically confirmed patients suffering from SCA (SCA1 - 12, SCA2 - 9, and SCA3 - 10) were studied. High resolution T1-weighted 3-Dimensional Magnetic Resonance Images of 31 patients were analyzed using the optimized VBM procedure. RESULTS In all the three SCAs there was a significant loss of gray matter in both cerebellar hemispheres and vermis. Vermian atrophy was more pronounced in SCA3, while SCA1 and SCA2 had significant white matter atrophy. Pontine white matter atrophy was more pronounced in SCA2. In SCA1, the severity of ataxia strongly correlated with the degree of gray matter atrophy in cerebellar hemispheres. The duration of symptoms and lengths of CAG repeats had no correlation with the degree of atrophy. CONCLUSIONS This study showed that the different subtypes of SCAs may have morphometric differences in the cerebellum, brainstem and the supratentorial structures.

SCA-LSVD: a repeat-oriented locus-specific variation database for genotype to phenotype correlations in spinocerebellar ataxias.

Repeat expansion has been implicated in 10 out of 17 candidate genes identified for autosomal dominant cerebellar ataxias (ADCAs)—commonly referred as spinocerebellar ataxias (SCAs). Though genetically distinct, the SCAs share a large number of features that confound their clinical classification. In addition, there is a difference in the prevalence and phenotypic expression of ataxias between different ethnic groups. We have created a new SCA‐locus‐specific variation database (LSVD) that aims to catalog and integrate information on SCAs associated with trinucleotide repeat expansion (SCA1, SCA 2, SCA 3, SCA 6, SCA 7, SCA 8, SCA 12, SCA 17, Friedreichs ataxia [FRDA], and dentatorubral‐pallidoluysian atrophy [DRPLA]) from all over the world. The database has been developed using the Leiden Open (source) Variation Database (LOVD) software (Leiden University Medical Center, Leiden, the Netherlands). The database houses detailed information on clinical features, such as age and symptom at onset, mode of inheritance, and genotype information, pertaining to the SCA patients from more than 400 families across India. All the compiled genotype data conforms to the HGVS Nomenclature guidelines. This would be a very useful starting point for understanding the molecular correlates of phenotypes in ataxia—a multilocus disease in which related molecular mechanisms converge to overlapping phenotypes. The database is accessible online at http://miracle.igib.res.in/ataxia. Hum Mutat 30:1–6, 2009.

Oncogenic KIAA1549-BRAF fusion with activation of the MAPK/ERK pathway in pediatric oligodendrogliomas

Pediatric oligodendrogliomas (pODGs) are rare central nervous system tumors, and comparatively little is known about their molecular pathogenesis. Co-deletion of 1p/19q; and IDH1, CIC, and FUBP1 mutations, which are molecular signatures of adult oligodendrogliomas, are extremely rare in pODGs. In this report, two pODGs, one each of grade II and grade III, were evaluated using clinical, radiological, histopathologic, and follow-up methods. IDH1, TP53, CIC, H3F3A, and BRAF-V600 E mutations were analyzed by Sanger sequencing and immunohistochemical methods, and 1p/19q co-deletion was analyzed by fluorescence in situ hybridization. PDGFRA amplification, BRAF gain, intragenic duplication of FGFR-TKD, and KIAA1549-BRAF fusion (validated by Sanger sequencing) were analyzed by real-time reverse transcription PCR. Notably, both cases showed the oncogenic KIAA1549_Ex15-BRAF_Ex9 fusion transcript. Further, immunohistochemical analysis showed activation of the MAPK/ERK pathway in both of these cases. However, neither 1p/19q co-deletion; IDH1, TP53, CIC, H3F3A, nor BRAF-V600 E mutation; PDGFRA amplification; BRAF gain; nor duplication of FGFR-TKD was identified. Overall, this study highlights that pODGs can harbor the KIAA1549-BRAF fusion with aberrant MAPK/ERK signaling, and there exists an option of targeting these pathways in such patients. These results indicate that pODGs with the KIAA1549-BRAF fusion may represent a subset of this rare tumor that shares molecular and genetic features of pilocytic astrocytomas. These findings will increase our understanding of pODGs and may have clinical implications.

North and South Indian Populations Share a Common Ancestral Origin of Friedreich's Ataxia but Vary in Age of GAA Repeat Expansion

Friedreichs ataxia (FRDA) is caused by expansion of GAA repeats in the frataxin (FXN) gene on chromosome 9q13‐q21.1. We analysed the origin of FRDA in 21 North Indian (NI) and eight South Indian (SI) families using five single nucleotide polymorphisms (SNPs) and a microsatellite marker spanning the GAA repeats. The NI and SI families were derived from Indo‐European and Dravidian linguistic backgrounds respectively. The frequency of large normal (LNs) alleles of the GAA repeat correlate with the overall lower prevalence of FRDA in India compared to the European population. All of the expanded alleles in the Indian population share a common core haplotype suggesting a founder effect. The expanded alleles in the NI population demonstrate more similarity to those of Europeans in terms of age of GAA repeat expansion (15975 ± 2850 years) and association of LNs with expanded alleles. FRDA seems to have been introduced recently in the South Indian population since the average estimated age of the mutation in SI is 5425 ± 1750 years and unlike NI some of the haplotypes of LNs are not associated with the expanded alleles.

Synthetic transcription elongation factors license transcription across repressive chromatin

Chemical control of transcription Friedreichs ataxia, a devastating neurodegenerative disease with no effective therapy, is caused by an expansion of intronic repeats and hence a reduced expression of the FXN gene. Erwin et al. synthesized a molecule that specifically targets the expanded repressive repeats. This molecule thereby licenses productive transcription elongation and restores FXN expression to normal levels. In the future, similar interventions may be effective in a diverse array of diseases caused by unstable expansions in microsatellite repeats. Science, this issue p. 1617 A synthetic molecule can surmount barriers to transcription elongation at repressive microsatellite repeats that cause Friedreich’s ataxia. The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich’s ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.

Infantile Onset Spinocerebellar Ataxia 2 (SCA2) A Clinical Report With Review of Previous Cases

Autosomal dominant cerebellar ataxia type I is a heterogenous group of spinocerebellar ataxias with variable neurologic presentations, with age of onset varying from infancy to adulthood. Autosomal dominant cerebellar ataxia type I is composed mainly of 3 prevalent spinocerebellar ataxia types with different pathogenic loci, specifically spinocerebellar ataxia 1 (6p24-p23), spinocerebellar ataxia 2 (12q24.1), and spinocerebellar ataxia 3 (14q32.1). The shared pathogenic mutational event is the expansion of the CAG repeat that results in polyglutamine extended stretches in the encoded proteins. CAG repeat disorders generally show the phenomenon of anticipation, which is more often associated with paternal transmission. In this report, we describe a patient with infantile-onset spinocerebellar ataxia type 2 (∼320 CAG repeat) who inherited the disease from his father (47 CAG repeats). We have summarized the clinical, neuroimaging, electroencephalographic (EEG), and molecular data of previous cases and attempt to highlight the most consistent findings. Our intent is to help treating clinicians to suspect this disorder and to offer timely genetic counseling for a currently potentially untreatable disorder.

Novel mutations in typical and atypical genetic loci through exome sequencing in autosomal recessive cerebellar ataxia families.

Nearly a thousand mutations mapping to 60 different loci have been identified in cerebellar ataxias. However, almost 50% of the cases remain genetically uncharacterized and there is a difference in prevalence as well as in the phenotypic spectrum of ataxia among various geographical regions. This poses a challenge for setting up a genetic panel for screening ataxia. In our ataxic cohort of 1014 families, 61% are genetically uncharacterized (UC). We investigated the potential of whole exome sequencing in conjunction with homozygosity mapping (HM) to delineate the genetic defects in three uncharacterized families with recessive inheritance each manifesting some unusual phenotype: (i) infantile onset ataxia with hearing loss (IOAH), (ii) Juvenile onset cerebellar ataxia with seizures (JCS) and (iii) Friedreich ataxia‐like (FA‐like). We identified a novel missense mutation in c10orf2 in the family with IOAH, compound heterozygous mutations in CLN6 in the family with JCS and a homozygous frame‐shift mutation in SACS in the FA‐like patient. Phenotypes observed in our families were concordant with reported phenotypes of known mutations in the same genes thus obviating the need for functional validation. Our study revealed novel variations in three genes, c10orf2, CLN6, and SACS, that have so far not been reported in India. This study also demonstrates the utility of whole exome screening in clinics for early diagnosis.

Genetic alterations related to BRAF‐FGFR genes and dysregulated MAPK/ERK/mTOR signaling in adult pilocytic astrocytoma

Pilocytic astrocytomas occur rarely in adults and show aggressive tumor behavior. However, their underlying molecular‐genetic events are largely uncharacterized. Hence, 59 adult pilocytic astrocytoma (APA) cases of classical histology were studied (MIB‐1 LI: 1%–5%). Analysis of BRAF alterations using qRT‐PCR, confirmed KIAA1549‐BRAF fusion in 11 (19%) and BRAF‐gain in 2 (3.4%) cases. BRAF‐V600E mutation was noted in 1 (1.7%) case by sequencing. FGFR1‐mutation and FGFR‐TKD duplication were seen in 7/59 (11.9%) and 3/59 (5%) cases, respectively. Overall 36% of APAs harbored BRAF and/or FGFR genetic alterations. Notably, FGFR related genetic alterations were enriched in tumors of supratentorial region (8/25, 32%) as compared with other locations (P = 0.01). The difference in age of cases with FGFR1‐mutation (Mean age ± SD: 37.2 ± 15 years) vs. KIAA1549‐BRAF fusion (Mean age ± SD: 25.1 ± 4.1 years) was statistically significant (P = 0.03). Combined BRAF and FGFR alterations were identified in 3 (5%) cases. Notably, the cases with more than one genetic alteration were in higher age group (Mean age ± SD: 50 ± 12 years) as compared with cases with single genetic alteration (Mean age ± SD: 29 ± 10; P = 0.003). Immunopositivity of p‐MAPK/p‐MEK1 was found in all the cases examined. The pS6‐immunoreactivity, a marker of mTOR activation was observed in 34/39 (87%) cases. Interestingly, cases with BRAF and/or FGFR related alteration showed significantly lower pS6‐immunostatining (3/12; 25%) as compared with those with wild‐type BRAF and/or FGFR (16/27; 59%) (P = 0.04). Further, analysis of seven IDH wild‐type adult diffuse astrocytomas (DA) showed FGFR related genetic alterations in 43% cases. These and previous results suggest that APAs are genetically similar to IDH wild‐type adult DAs. APAs harbor infrequent BRAF alterations but more frequent FGFR alterations as compared with pediatric cases. KIAA1549‐BRAF fusion inversely correlates with increasing age whereas FGFR1‐mutation associates with older age. Activation of MAPK/ERK/mTOR signaling appears to be an important oncogenic event in APAs and may be underlying event of aggressive tumor behavior. The findings provided a rationale for potential therapeutic advantage of targeting MAPK/ERK/mTOR pathway in APAs.

Study of β-catenin and BRAF alterations in adamantinomatous and papillary craniopharyngiomas: mutation analysis with immunohistochemical correlation in 54 cases

Craniopharyngiomas (CP) are rare benign epithelial tumors, with two histological variants, namely the adamantinomatous variant (ACP) and the rarer papillary variant (PCP). They are locally infiltrative and surgically challenging tumors with severe long term morbidity. CTNNB1 mutations with β-catenin immunopositivity and BRAFV600E mutations with anti-VE immunopositivity have been recently described in ACPs and PCPs respectively. We aimed to study BRAF and CTNNB1 gene mutations in CPs operated at our institute, and correlate it with clinicopathological parameters including histopathology and immunohistochemistry (IHC) for proteins VE-1 and β-catenin. A total of 54 CPs diagnosed over 3-year duration were included. IHC for β-catenin and VE-1 proteins, and Sanger sequencing for CTNNB1 (exon 3) and BRAF (exon 15) genes were performed. CTNNB1 mutations were identified in 63% (27/43) of ACPs while nuclear immunopositivity for β-catenin was observed in 79% (34/43) of them. Seven ACPs showed β-catenin immunopositivity in the absence of mutations. BRAFV600E (p.Val600Glu) mutations were observed in 57% of PCPs (4/7), while cytoplasmic immunopositivity for anti-VE1 antibody was observed only in 43% of PCPs (3/7), all of which also harboured BRAFV600E mutations. The mutations and IHC staining patterns of ACPs and PCPs were non-overlapping. Four cases with uncertain histological pattern could be subcategorised into specific variants only following mutation analysis/IHC. The identification of hallmark molecular signatures in the two CP variants holds promise for alternate improved treatment modalities, emphasizing the need for sub-categorization in routine histopathology reporting. IHC for β-catenin and targeted sequencing for BRAFV600E serve as useful adjuncts.