Ery Kus Dwianingsih

A Japanese child with asymptomatic elevation of serum creatine kinase shows PTRF-CAVIN mutation matching with congenital generalized lipodystrophy type 4.

Congenital generalized lipodystrophy (CGL), characterized by generalized absence of adipose tissue, has heterogeneous causes. Recently, a novel type of CGL complicated by muscular dystrophy was categorized as CGL4 caused by PTRF-CAVIN deficiency. However, it is unknown whether CGL4 exhibits clinical abnormalities during the infantile period. Here, we describe the youngest Japanese case of CGL4-a Japanese girl with asymptomatic high serum creatine kinase (CK) levels at 3months old. She was referred to our hospital at 5months of age because of her elevated serum CK (2528IU/L). Generalized absence of adipose tissue was first recognized at 2years of age. Mutation analysis of genes known to be responsible for CGL1-3 failed to disclose any abnormalities. Instead, analysis of the PTRF-CAVIN gene encoding PTRF-CAVIN revealed compound heterozygous mutations, one allele contained an insertion (c.696_697insC) and the other allele harbored a novel nonsense mutation (c.512C>A). Our patient had low serum leptin and adiponectin levels and insulin resistance. Pathological studies on biopsied muscle disclosed mild dystrophic change and highly reduced expression of PTRF-CAVIN. It was concluded that our PTRF-CAVIN deficient patient showed not only CGL but also asymptomatic elevation of serum CK because of her mild muscle dystrophic change.

A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy

Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease, is mostly caused by exon deletion mutations in the DMD gene. The reading frame rule explains that out-of-frame deletions lead to muscle dystrophin deficiency in DMD. In outliers to this rule, deletion junction sequences have never previously been explored as splicing modulators. In a Japanese case, we identified a single exon 45 deletion in the patient’s DMD gene, indicating out-of-frame mutation. However, immunohistochemical examination disclosed weak dystrophin signals in his muscle. Reverse transcription-PCR amplification of DMD exons 42 to 47 revealed a major normally spliced product with exon 45 deletion and an additional in-frame product with deletion of both exons 44 and 45, indicating upstream exon 44 skipping. We considered the latter to underlie the observed dystrophin expression. Remarkably, the junction sequence cloned by PCR walking abolished the splicing enhancer activity of the upstream intron in a chimeric doublesex gene pre-mRNA in vitro splicing. Furthermore, antisense oligonucleotides directed against the junction site counteracted this effect. These indicated that the junction sequence was a splicing silencer that induced upstream exon 44 skipping. It was strongly suggested that creation of splicing regulator is a modifier of dystrophinopathy.

Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers

BackgroundDuchenne muscular dystrophy, a fatal muscle-wasting disease, is characterized by dystrophin deficiency caused by mutations in the dystrophin gene. Skipping of a target dystrophin exon during splicing with antisense oligonucleotides is attracting much attention as the most plausible way to express dystrophin in DMD. Antisense oligonucleotides have been designed against splicing regulatory sequences such as splicing enhancer sequences of target exons. Recently, we reported that a chemical kinase inhibitor specifically enhances the skipping of mutated dystrophin exon 31, indicating the existence of exon-specific splicing regulatory systems. However, the basis for such individual regulatory systems is largely unknown. Here, we categorized the dystrophin exons in terms of their splicing regulatory factors.ResultsUsing a computer-based machine learning system, we first constructed a decision tree separating 77 authentic from 14 known cryptic exons using 25 indexes of splicing regulatory factors as decision markers. We evaluated the classification accuracy of a novel cryptic exon (exon 11a) identified in this study. However, the tree mislabeled exon 11a as a true exon. Therefore, we re-constructed the decision tree to separate all 15 cryptic exons. The revised decision tree categorized the 77 authentic exons into five groups. Furthermore, all nine disease-associated novel exons were successfully categorized as exons, validating the decision tree. One group, consisting of 30 exons, was characterized by a high density of exonic splicing enhancer sequences. This suggests that AOs targeting splicing enhancer sequences would efficiently induce skipping of exons belonging to this group.ConclusionsThe decision tree categorized the 77 authentic exons into five groups. Our classification may help to establish the strategy for exon skipping therapy for Duchenne muscular dystrophy.

The Polymerase Chain Reaction in Diagnosis of Small B-Cell Non-Hodgkin Lymphomas.

BACKGROUND Small B-cell non-Hodgkins lymphoma (NHL) is difficult to be distinguished from non-neoplastic reactive processes using conventional haematoxylin-eosin (HE) staining due to different interpretations among pathologists with diagnosis based on morphologic features. Ancillary examinations such as immunohistochemical (IHC) staining are essential. However, negative or doubtful results are still sometimes obtained due to unsatisfactory tissue processing or IHC technique. The polymerase chain reaction (PCR) as a molecular diagnostic technique is very sensitive and specific. Clonality detection of heavy chain immunoglobulin (IgH) gene rearrangement has been widely used to establish diagnosis of B-cell NHL. AIMS To elaborate interobserver variation in small B-cell NHL diagnosis based on morphologic features only and to confirm sensitivity and specificity of the PCR technique as an ancillary method. MATERIALS AND METHODS A toptal of 28 samples of small B cell NHL and suspicious lymphoma were interpreted by 3 pathologists in Sardjito General Hospital based on their morphology only. The reliability of assessment and the coefficient of interobserver agreement were calculated by Fleiss kappa statistics. Interpretation results were confirmed with IHC staining (CD20, CD3, Bcl2). PCR was performed to analyze the clonality of IgH gene rearrangement. RESULTS Interobserver agreement in morphologic evalution of small B cell NHL and chronic lymphadenitis revealed kappa coefficient 0.69 included in the substantial agreement category. The cases were divided into 3 groups based on morphology and IHC results; lymphoma, reactive process and undetermined group. PCR analysis showed 90% sensitivity and 60% specificity. CONCLUSIONS The present study revealed a substantial agreement among pathologists in small B-cell NHL diagnosis. For difficult cases, PCR is useful as complementary method to morphologic and IHC examinations to establish definitive diagnosis.

Prognostic Value of Lymphangiogenesis Determinants in Luminal and Non-luminal Breast Carcinomas

Background: Breast carcinomas (BCs) are sub-classified according to the molecular characteristics into luminal and non-luminal subtypes that clinically show different biological behavior, treatment and prognosis. BCs spread primarily through lymphatic vessels using cascade processes of lymphagiogenesis in which VEGF-C plays an important role during lymph node metastasis. Prognostic value of VEGF-C in luminal and non-luminal BC is still unclear and has not been studied thoroughly to clarify and define prognosis and therapeutic monitoring. Aim: To define the prognostic value of lymphangiogenesis on survival rates of luminal and non-luminal subtypes BC. Materials and Methods: This study applied prospective cohort design, using 130 patients of invasive duct carcinoma of the breast, stage I-IIIA, from Sardjito General Hospital, Indonesia and subsequent longitudinal follow-up. Immunohistochemical staining was carried out using anti-ER, -PR, -Her-2, VEGF-C, VEGFR-3 and D2-40 antibodies. The related clinicopathologic characteristics of BC patients and lymphangiogenesis determinants, including VEGF-C expression, were statistically analyzed. Results: In non-luminal BC subtypes, VEGF-C expression (HR=0.04; 95% CI=0.01-0.41), lymph node metastasis (HR=0.14; 95% CI=0.04-0.55) and stage (HR=0.30; 95% CI= 0.02-0.76) were determined as independent prognostic factors on survival rates. However, the lymphangiogenesis determinants were not associated with the survival rates of luminal BC subtypes. Conclusion: This study suggested that lymphangiogenesis affects survival rates of non-Luminal subtype rather than the luminal subtypes of BC.

Prognostic Value of Clinicopathological Factors for Indonesian Breast Carcinomas of Different Molecular Subtypes

Background: Breast carcinoma (BC) is a heterogeneous disease due to its different molecular profiles i.e. luminal (luminal A and luminal B) and non-luminal (HER2 positive and triple negative) subtypes. Prognostic value of clinicopathological factors of Indonesian BC of different molecular subtypes has never been reported previously. This study aims to elaborate prognostic impacts on Indonesian BCs focusing on separate molecular subtypes. Methods: A hundred and fifty cases of invasive BC, stage I-IIIA, in Sardjito Hospital, Indonesia, were stained using anti ER, PR, HER2 and Ki-67 antibodies. Survival and prognostic values were statistically analyzed. Results: Compared to the luminal subtypes, the non-luminal subtypes demonstrated higher proportions of intermediate-to-high grade, stage IIIA, positive lymph node infiltration and mortality. The triple negative subtype was typically intermediate-to-high grade, stage IIIA and with a high relative death risk. Luminal A lesions were characteristically low grade, stage I-II and less likely to cause death. Conclusion: In non-luminal BC, staging and lymph node metastasis are independent prognostic factors for survival in HER2 positive and triple negative subtypes, respectively. In luminal BC, clinicopathological factors demonstrated no influence on survival. This study suggests that staging and lymph node metastasis are correlated with survival in non-luminal Indonesian BCs.

T.P.29 Categorization of 77 dystrophin exons into five groups by a decision tree using indexes of splicing regulatory factors

Abstract Duchenne muscular dystrophy (DMD), a fatal muscle-wasting disease, is characterized by dystrophin deficiency caused by mutations in the dystrophin gene. Skipping of a target dystrophin exon during splicing with antisense oligonucleotides is attracting much attention as the most plausible way to express dystrophin in DMD. Antisense oligonucleotides (AOs) have been designed against splicing regulatory sequences such as splicing enhancer sequences of target exons. Recently, we reported that a chemical kinase inhibitor specifically enhances the skipping of mutated dystrophin exon 31, indicating the existence of exon-specific splicing regulatory systems. However, the basis for such individual regulatory systems is largely unknown. Here, we categorized the dystrophin exons in terms of their splicing regulatory factors. Using a computer-based machine learning system, we first constructed a decision tree separating 77 authentic from 14 known cryptic exons using 26 indexes of splicing regulatory factors as decision markers. We evaluated the classification accuracy of a novel cryptic exon (exon 11a) identified in this study. However, the tree mislabeled exon 11a as a true exon. Therefore, we re-constructed the decision tree to separate all 15 cryptic exons. The revised decision tree categorized the 77 authentic exons into five groups. Furthermore, all nine disease-associated novel exons were successfully categorized as exons, validating the decision tree. One group, consisting of 30 exons, was characterized by a high density of exonic splicing enhancer sequences. This suggests that AOs targeting splicing enhancer sequences would efficiently induce skipping of exons belonging to this group. Our classification may help to establish the strategy for exon skipping therapy for Duchenne muscular dystrophy.