Capsule Summary

In Kawasaki Disease patients, the authors show associations between high-dose intravenous immunoglobulin (IVIG) response and a polymorphism in the FCγRIIB. This provides basis for defining the IVIG regulatory mechanisms and pharmacogenomic approach to IVIG therapy.

We studied a man with distal hypospadias, partial anomalous pulmonary venous return, mild limb-length inequality and a balanced translocation involving chromosomes 9 and 13. To gain insight into the etiology of his birth defects, we mapped the translocation breakpoints by high-resolution comparative genomic hybridization (CGH), using chromosome 9- and 13-specific tiling arrays to analyze genetic material from a spontaneously aborted fetus with unbalanced segregation of the translocation. The chromosome 13 breakpoint was ∼400 kb away from the nearest gene, but the chromosome 9 breakpoint fell within an intron of Basonuclin 2 (BNC2), a gene that encodes an evolutionarily conserved nuclear zinc-finger protein. The BNC2/Bnc2 gene is abundantly expressed in developing mouse and human periurethral tissues. In all, 6 of 48 unrelated subjects with distal hypospadias had nine novel nonsynonymous substitutions in BNC2, five of which were computationally predicted to be deleterious. In comparison, two of 23 controls with normal penile urethra morphology, each had a novel nonsynonymous substitution in BNC2, one of which was predicted to be deleterious. Bnc2−/− mice of both sexes displayed a high frequency of distal urethral defects; heterozygotes showed similar defects with reduced penetrance. The association of BNC2 disruption with distal urethral defects and the gene's expression pattern indicate that it functions in urethral development.

Objectives

We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients.

Background

CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction.

Methods

In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription–polymerase chain reaction.

Results

Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP.

Conclusions

ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), whereas three other genes (actin, lamin A/C, and desmin) cause autosomal dominant DCM; seven other loci for autosomal dominant DCM have been mapped but the genes have not been identified. Hypothesizing that DCM is a disease of the cytoskeleton and sarcolemma, we have focused on candidate genes whose products are found in these structures. Here we report the screening of the human δ-sarcoglycan gene, a member of the dystrophin-associated protein complex, by single-stranded DNA conformation polymorphism analysis and by DNA sequencing in patients with DCM. Mutations affecting the secondary structure were identified in one family and two sporadic cases, whereas immunofluorescence analysis of myocardium from one of these patients demonstrated significant reduction in δ-sarcoglycan staining. No skeletal muscle disease occurred in any of these patients. These data suggest that δ-sarcoglycan is a disease-causing gene responsible for familial and idiopathic DCM and lend support to our “final common pathway” hypothesis that DCM is a cytoskeletalopathy.

Objective

Evaluate the outcome and prevalence of viral endomyocardial infection after cardiac transplantation.

Background

Viral myocardial infection causes heart failure, but its role after cardiac transplantation is unclear. We hypothesized that viral infection of the cardiac allograft reduces graft survival.

Methods

Between 6/1999 and 11/2004, 94 pediatric cardiac transplant patients were screened for the presence of viral genome in serial endomyocardial biopsies (EMBs) using PCR assays. Graft loss, advanced transplant coronary artery disease (TCAD) and acute rejection (AR) were compared in the PCR-positive (PCR+) (n=37) and PCR-negative (PCR−) (n=57) groups, using time dependent Kaplan-Meier and Cox regression analyses. From 11/2002 to 11/2004, intravenous immunoglobulin therapy (IVIG) was administered to patients with PCR+ EMBs. The outcomes of the IVIG-treated, PCR+ patients (n=20) were compared with IVIG-untreated, PCR+ patients (n=17).

Results

Viral genomes were detected in EMBs from 37 (39%) patients; parvovirus B19, adenovirus, & EBV were the most common. The `PCR+ group' (n=37, 25% graft loss at 2.4 years) had decreased graft survival (p<0.001) compared to the `PCR- group' (n=57, 25% graft loss at 8.7 years) and developed advanced TCAD prematurely (p=0.001). The number of AR episodes was similar in both groups. On multivariate analysis, presence of viral genome was an independent risk factor for graft loss (relative risk 4.2, p=0.015). The time to advanced TCAD after becoming PCR+ was longer in the IVIG-treated patients (p=0.03), with a trend towards improved graft survival (p=0.06).

Conclusions

Viral endomyocardial infection is an independent predictor of graft loss in pediatric cardiac transplant recipients. This effect appears to be mediated through premature development of advanced TCAD. IVIG therapy in this subgroup may improve survival and merits further investigation.

Background

Detection of viral genome in rejecting cardiac transplant patients has been reported, with coxsackievirus and adenovirus causing premature graft failure. Recently, parvovirus B19 (PVB19) genome in myocardial samples has been increasingly reported but its role in cardiac pathology and effect on transplant graft survival are unknown. The objectives were to determine if changes in the viruses identified in the myocardium represent an epidemiologic shift in viral myocardial disease and whether PVB19 adversely affects transplant graft survival.

Methods

From 9/2002 to 12/2005, 99 children (3 weeks-18 years) with heart transplants had endomyocardial biopsies evaluated for the presence of viral genome utilizing nested PCR. Cellular rejection was assessed by histology of biopsies, while transplant coronary artery disease (TCAD) was diagnosed by coronary angiography or histopathology.

Results

Seven hundred biopsies were evaluated from 99 patients; 121 biopsies had viral genome with 100 (82.6%) positive for PVB19, 24 for Epstein-Barr virus (EBV; 7 positive for PVB19 and EBV), 3 for CMV and 1 for adenovirus. Presence of PVB19 genome did not correlate with rejection score, nor did higher viral copy number. Children with persistent PVB19 infection (>6 months; n=20), had early development of advanced TCAD (p<0.001).

Conclusions

PVB19 is currently the predominant virus detected in heart transplant surveillance biopsies, possibly representing an epidemiologic shift. While cellular rejection does not correlate with the presence or quantity of PVB19 genome in the myocardium, children with chronic PVB19 infection have increased risk for earlier TCAD, supporting the hypothesis that PVB19 negatively affects graft survival.

Objectives

Four variants (K60N, Q128R, G202R and A592E) in the nebulette gene (NEBL) were identified in patients with dilated cardiomyopathy (DCM) and endocardial fibroelastosis (EFE). We sought to determine if these mutations cause cardiomyopathy.

Background

Nebulette aligns thin filaments and connects them with the myocardial Z-disk.

Methods

We produced transgenic mice with cardiac-restricted over-expression of human wild-type (WT) or mutant nebulette. Chimera and transgenic mice were examined at 4, 6 and 12 months of age by echocardiography and cardiac MRI. The hearts from embryos and adult mice were assessed by histopathologic, immunohistochemical, ultrastructural and protein analyses. Rat H9C2 cardiomyoblasts with transient expression of nebulette underwent cyclic mechanical strain.

Results

We identified lethal cardiac structural abnormalities in mutant embryonic hearts (K60N and Q128R). Founders of the mutant mice lines developed DCM with severe heart failure. An irregular localization pattern for nebulette and impaired desmin expression was noted in the proband and chimera Q128R mice. Mutant G202R and A592E mice exhibited left ventricular dilation and impaired cardiac function accompanied with the specific changes in I-band or Z-disk proteins by 6 months of age, respectively. The mutations modulated distribution of nebulette in the sarcomere and the Z-disks during stretch of H9C2 cells.

Conclusions

NEBL is a new susceptibility gene for EFE and DCM. Different mutations in nebulette trigger specific mechanisms converging to a common pathological cascade leading to EFE and DCM.