Karen M. Chisholm

Mutations in mitochondrial histidyl tRNA synthetase HARS2 cause ovarian dysgenesis and sensorineural hearing loss of Perrault syndrome

Perrault syndrome is a genetically heterogeneous recessive disorder characterized by ovarian dysgenesis and sensorineural hearing loss. In a nonconsanguineous family with five affected siblings, linkage analysis and genomic sequencing revealed the genetic basis of Perrault syndrome to be compound heterozygosity for mutations in the mitochondrial histidyl tRNA synthetase HARS2 at two highly conserved amino acids, L200V and V368L. The nucleotide substitution creating HARS2 p.L200V also created an alternate splice leading to deletion of 12 codons from the HARS2 message. Affected family members thus carried three mutant HARS2 transcripts. Aminoacylation activity of HARS2 p.V368L and HARS2 p.L200V was reduced and the deletion mutant was not stably expressed in mammalian mitochondria. In yeast, lethality of deletion of the single essential histydyl tRNA synthetase HTS1 was fully rescued by wild-type HTS1 and by HTS1 p.L198V (orthologous to HARS2 p.L200V), partially rescued by HTS1 p.V381L (orthologous to HARS2 p.V368L), and not rescued by the deletion mutant. In Caenorhabditis elegans, reduced expression by RNAi of the single essential histydyl tRNA synthetase hars-1 severely compromised fertility. Together, these data suggest that Perrault syndrome in this family was caused by reduction of HARS2 activity. These results implicate aberrations of mitochondrial translation in mammalian gonadal dysgenesis. More generally, the relationship between HARS2 and Perrault syndrome illustrates how causality may be demonstrated for extremely rare inherited mutations in essential, highly conserved genes.

Mutations in the DBP-Deficiency Protein HSD17B4 Cause Ovarian Dysgenesis, Hearing Loss, and Ataxia of Perrault Syndrome

Perrault syndrome is a recessive disorder characterized by ovarian dysgenesis in females, sensorineural deafness in both males and females, and in some patients, neurological manifestations. No genes for Perrault syndrome have heretofore been identified. A small family of mixed European ancestry includes two sisters with well-characterized Perrault syndrome. Whole-exome sequencing of genomic DNA from one of these sisters revealed exactly one gene with two rare functional variants: HSD17B4, which encodes 17beta-hydroxysteroid dehydrogenase type 4 (HSD17B4), also known as D-bifunctional protein (DBP). HSD17B4/DBP is a multifunctional peroxisomal enzyme involved in fatty acid beta-oxidation and steroid metabolism. Both sisters are compound heterozygotes for HSD17B4 c.650A>G (p.Y217C) (maternal allele) and HSB17B4 c.1704T>A (p.Y568X) (paternal allele). The missense mutation is predicted by structural analysis to destabilize the HSD17B4 dehydrogenase domain. The nonsense mutation leads to very low levels of HSD17B4 transcript. Expression of mutant HSD17B4 protein in a compound heterozygote was severely reduced. Mutations in HSD17B4 are known to cause DBP deficiency, an autosomal-recessive disorder of peroxisomal fatty acid beta-oxidation that is generally fatal within the first two years of life. No females with DBP deficiency surviving past puberty have been reported, and ovarian dysgenesis has not previously been associated with this illness. Six other families with Perrault syndrome have wild-type sequences of HSD17B4. These results indicate that Perrault syndrome and DBP deficiency overlap clinically; that Perrault syndrome is genetically heterogeneous; that DBP deficiency may be underdiagnosed; and that whole-exome sequencing can reveal critical genes in small, nonconsanguineous families.

Detection of long non-coding RNA in archival tissue: correlation with polycomb protein expression in primary and metastatic breast carcinoma.

A major function of long non-coding RNAs (lncRNAs) is regulating gene expression through changes in chromatin state. Experimental evidence suggests that in cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an occupancy pattern resembling that of the embryonic state. We have previously demonstrated that the expression level of lncRNA in the HOX locus, including HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in formalin-fixed paraffin-embedded breast cancer tissues in a high throughput tissue microarray format to correlate expression with clinicopathologic features. Though overall EZH2 and HOTAIR expression levels were highly correlated, the subset of cases with strong HOTAIR expression correlated with ER and PR positivity, while the subset of cases with strong EZH2 expression correlated with an increased proliferation rate, ER and PR negativity, HER2 underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and EZH2 had increased expression in the metastatic carcinomas. This is the first study to show that RNA in situ hybridization of formalin fixed paraffin-embedded clinical material can be used to measure levels of long non-coding RNAs. This approach offers a method to make observations on lncRNAs that may influence the cancer epigenome in a tissue-based technique.

β -Adrenergic receptor expression in vascular tumors

Propranolol has recently emerged as an effective therapy for infantile hemangiomas causing regression. The β-adrenergic receptor (AR) antagonist is thought to cause vasoconstriction by its effect on nitric oxide, block angiogenesis by its effect on vascular endothelial growth factor (VEGF), and induce apoptosis. In a prior report, we identified expression of β2-AR (B2-AR) and its phosphorylated form (B2-ARP) in a case of infantile hemangioma that responded to propranolol treatment. We now explore the expression of βARs on a variety of vascular lesions utilizing a tissue microarray containing 141 lesions, including infantile hemangiomas, angiosarcomas, hemangiomas, hemangioendotheliomas, and various vascular malformations. The array was immunostained for B2-AR, B2-ARP, and β3-AR (B3-AR), and the results scored for the intensity of endothelial cell expression as negative, weak positive, or strong positive. All phases of infantile hemangiomas had strong expression of all three receptors, with the exception of only weak expression of B2-ARP in the proliferative phase infantile hemangioma. Strong expression of all three receptors was present in many hemangiomas, hemangioendotheliomas, and vascular malformations. Absent to weak expression of all three receptors was seen in glomus tumor, hobnail hemangioendothelioma, pyogenic granuloma, and reactive vascular proliferations. This is the first study to report β-AR expression in a variety of vascular lesions. Although immunohistochemical expression of the receptors does not necessarily indicate that similar pathways of responsiveness to β-blockade are present, it does raises the possibility that β-blockade could potentially affect apoptosis and decrease responsiveness to VEGF. Additional study is warranted, as therapeutic options are limited for some patients with these lesions.

Expression profiles of MYC protein and MYC gene rearrangement in lymphomas.

MYC translocations are a defining feature of Burkitt lymphoma and a group of diffuse large B-cell lymphoma (DLBCL) with inferior outcome. However, the clinical relevance of MYC gene rearrangement and its relationship with MYC protein expression has not been well characterized in lymphomas. Tissue microarrays containing 1214 lymphomas were successfully evaluated by immunohistochemistry using anti-MYC clone Y69 and a dual-color break-apart fluorescence in situ hybridization probe to detect MYC gene rearrangements. Aggressive B-cell lymphomas including Burkitt lymphoma and DLBCL showed the highest level of MYC protein staining defined as staining in >50% of lymphoma cells. A significant proportion of plasmablastic, B-lymphoblastic and T-lymphoblastic, and extranodal NK/T-cell lymphomas also showed staining in >50% of cells, whereas only occasional plasma cell myeloma, mantle cell lymphoma, and classical Hodgkin lymphoma showed a high level of staining. Small B-cell lymphomas, when positive, showed MYC protein in <50% of cells. In aggressive B-cell lymphomas, MYC rearrangement and MYC immunohistochemistry showed a high concordance rate; however, some DLBCL and all T-cell and NK-cell lymphomas with MYC protein expression lacked MYC gene rearrangements. Our results provide a baseline for MYC protein expression in lymphomas and indicate that its expression is not specific to lymphoma subtypes, cell lineage, or expected clinical behavior and is highly variable. In addition, MYC protein expression is not necessarily correlated with MYC gene rearrangements and suggests the need for caution in the interpretation of MYC immunohistochemistry in the differential diagnosis of lymphomas.

Correlation between metabolic tumor volume and pathologic tumor volume in squamous cell carcinoma of the oral cavity.

PURPOSE To explore the relationship between pathologic tumor volume and volume estimated from different tumor segmentation techniques on (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET) in oral cavity cancer. MATERIALS AND METHODS Twenty-three patients with squamous cell carcinoma of the oral tongue had PET-CT scans before definitive surgery. Pathologic tumor volume was estimated from surgical specimens. Metabolic tumor volume (MTV) was defined from PET-CT scans as the volume of tumor above a given SUV threshold. Multiple SUV thresholds were explored including absolute SUV thresholds, relative SUV thresholds, and gradient-based techniques. RESULTS Multiple MTVs were associated with pathologic tumor volume; however the correlation was poor (R(2) range 0.29-0.58). The ideal SUV threshold, defined as the SUV that generates an MTV equal to pathologic tumor volume, was independently associated with maximum SUV (p=0.0005) and tumor grade (p=0.024). MTV defined as a function of maximum SUV and tumor grade improved the prediction of pathologic tumor volume (R(2)=0.63). CONCLUSIONS Common SUV thresholds fail to predict pathologic tumor volume in head and neck cancer. The optimal technique that allows for integration of PET-CT with radiation treatment planning remains to be defined. Future investigation should incorporate biomarkers such as tumor grade into definitions of MTV.

A genomewide screen for suppressors of Alu-mediated rearrangements reveals a role for PIF1.

Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition.

Heme oxygenase-1 deficiency promotes the development of necrotizing enterocolitis-like intestinal injury in a newborn mouse model

Necrotizing enterocolitis (NEC) is typified by mucosal destruction, which subsequently can lead to intestinal necrosis. Prematurity, enteral feeding, and bacterial colonization are the main risk factors and, combined with other stressors, can cause increased intestinal permeability, injury, and an exaggerated inflammatory response. Heme oxygenase-1 (HO-1) mediates intestinal protection due to anti-inflammatory, antioxidative, and antiapoptotic effects of its products carbon monoxide, biliverdin, and bilirubin. This study investigates a possible role of HO-1 in the pathogenesis of NEC using a newborn mouse model. We induced NEC-like intestinal injury in 7-day-old HO-1 heterozygous (HO-1 Het, Hmox1(+/-)) and wild-type (Wt, Hmox1(+/+)) mice by gavage feeding and hypoxic exposures. Control (Con) pups of both genotypes were dam-fed. Intestines of HO-1 Het Con pups appeared predisposed to injury, with higher histological damage scores, more TUNEL-positive cells, and a significant reduction in muscularis externa thickness compared with Wt Con pups. The increase in HO activity after HO-1 induction by the substrate heme or by hypoxic stress was significantly impaired in HO-1 Het pups. After induction of intestinal injury, HO-1 Het pups displayed significantly higher NEC incidence (78 vs. 43%), mortality (83 vs. 54%), and median scores (2.5 vs. 1.5) than Wt NEC pups. PCR array analyses revealed increased expressions of IL-1β, P-selectin, matrix metallopeptidase 2, collagen type XVIII-α1, serpine 1, and others in NEC-induced HO-1 Het ileal and jejunal tissues. We conclude that a partial HO-1 deficiency promotes experimental NEC-like intestinal injury, possibly mediated by exaggerated inflammation and disruption in tissue repair.

Fetal thrombotic vasculopathy: significance in liveborn children using proposed society for pediatric pathology diagnostic criteria.

Fetal thrombotic vasculopathy (FTV) is a recently described placental diagnosis associated with adverse perinatal outcomes. The Society for Pediatric Pathology proposed criteria for grading; however, no study has evaluated the proposed thresholds or established standards for large-vessel lesions. Using the Society for Pediatric Pathology criteria of 2 or more foci of 15 or more avascular villi or villous stromal-vascular karyorrhexis to represent severe FTV, this study examines the outcomes of liveborn infants with placentas demonstrating severe or nonsevere distal villous FTV (DV-FTV) and large-vessel FTV (LV-FTV). Control placentas over the same 3-year period were selected with minimal findings. Electronic medical records were queried for birth data, infant laboratory values, morbidities, and neurological development. The 139 cases included 102 with DV-FTV and 94 with LV-FTV. Compared with 111 controls, the 52 severe DV-FTV cases were significantly associated with delivery for fetal indications and small placental weight. The children with severe DV-FTV were more likely to be born small for gestational age, have intracranial hemorrhage, coagulopathy, neurological impairment, growth retardation, and evidence of systemic thrombosis/vasculopathy. Compared with controls, the 67 cases with severe LV-FTV were associated with maternal preeclampsia, delivery for fetal indications, small placental weight, umbilical cord abnormalities, and small size per gestational age. The 45 cases of DV-FTV or LV-FTV not classified as severe had similar characteristics as those without any FTV. In conclusion, severe FTV does appear associated with neurological injury, whereas those with nonsevere lesions have similar rates of morbidities as controls.

Diagnosis of congenital CMV using PCR performed on formalin-fixed, paraffin-embedded placental tissue.

Congenital cytomegalovirus (CMV) infection may be asymptomatic until hearing loss manifests in childhood. Because diagnosis of congenital CMV requires viral detection within an infant’s first 21 days of life, CMV polymerase chain reaction (PCR) on formalin-fixed, paraffin-embedded (FFPE) placental tissue provides a unique opportunity to identify congenital exposure in cases in which CMV is not initially suspected. To assess the utility of this approach, a database of all CMV cultures performed from July 2001 to March 2012 was used to identify infants in whom urine CMV cultures were obtained within 100 days of life. Corresponding placentas were then identified through the pathology database. The database was also queried to identify placentas in which CMV immunohistochemical analysis had been performed. CMV PCR was positive in FFPE placental tissue from 100% (5/5) of cases in which the first urine culture collected before the first 21 days of life was positive. Placentas from 20 infants with negative CMV urine cultures were CMV PCR negative. Interestingly, CMV was detected in 12.5% (1/8) of placentas in which the first CMV-positive urine culture was collected after the first 21 days of life. Furthermore, 4% (1/26) of placentas with chronic villitis by histology (no urine cultures available) were CMV PCR positive. In the 10 CMV PCR-positive placentas, including 3 cases of fetal demise, CMV immunohistochemistry was positive in just 6 cases. These results suggest that the confirmation of CMV exposure in utero by PCR of FFPE placental tissue provides a useful adjunct to histologic evaluation and may identify infants requiring close clinical follow-up.