Monozygotic twins discordant for constitutive BRCA1 promoter methylation, childhood cancer and secondary cancer.

We describe monozygotic twins discordant for childhood leukemia and secondary thyroid carcinoma. We used bisulfite pyrosequencing to compare the constitutive promoter methylation of BRCA1 and several other tumor suppressor genes in primary fibroblasts. The affected twin displayed an increased BRCA1 methylation (12%), compared with her sister (3%). Subsequent bisulfite plasmid sequencing demonstrated that 13% (6 of 47) BRCA1 alleles were fully methylated in the affected twin, whereas her sister displayed only single CpG errors without functional implications. This between-twin methylation difference was also found in irradiated fibroblasts and untreated saliva cells. The BRCA1 epimutation may have originated by an early somatic event in the affected twin: approximately 25% of her body cells derived from different embryonic cell lineages carry one epigenetically inactivated BRCA1 allele. This epimutation was associated with reduced basal protein levels and a higher induction of BRCA1 after DNA damage. In addition, we performed a genome-wide microarray analysis of both sisters and found several copy number variations, i.e., heterozygous deletion and reduced expression of the RSPO3 gene in the affected twin. This monozygotic twin pair represents an impressive example of epigenetic somatic mosaicism, suggesting a role for constitutive epimutations, maybe along with de novo genetic alterations in recurrent tumor development.

A girl with an atypical form of ataxia telangiectasia and an additional de novo 3.14 Mb microduplication in region 19q12.

A 9-year-old girl born to healthy parents showed manifestations suggestive of ataxia telangiectasia (AT), such as short stature, sudden short bouts of horizontal and rotary nystagmus, a weak and dysarthric voice, rolling gait, unstable posture, and atactic movements. She did not show several cardinal features typical of AT such as frequent, severe infections of the respiratory tract. In contrast, she showed symptoms not generally related to AT, including microcephaly, profound motor and mental retardation, small hands and feet, severely and progressively reduced muscle tone with slackly protruding abdomen and undue drooling, excess fat on her upper arms, and severe oligoarthritis. A cranial MRI showed no cerebellar hypoplasia and other abnormalities. In peripheral blood samples she carried a de novo duplication of 3.14 Mb in chromosomal region 19q12 containing six annotated genes, UQCRFS1, VSTM2B, POP4, PLEKHF1, CCNE1, and ZNF536, and a de novo mosaic inversion 14q11q32 (96% of metaphases). In a saliva-derived DNA sample only the duplication in 19q12 was detected, suggesting that the rearrangements in blood lymphocytes were acquired. These findings reinforced the suspicion that she had AT. AT was confirmed by strongly elevated serum AFP levels, cellular radiosensitivity and two inherited mutations in the ATM gene (c.510_511delGT; paternal origin and c.2922-50_2940del69; maternal origin). This case suggest that a defective ATM-dependent DNA damage response may entail additional stochastic genomic rearrangements. Screening for genomic rearrangements appears indicated in patients suspected of defective DNA damage responses.

In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.

BACKGROUND/AIMS: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. METHODS: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. RESULTS: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca(2+) release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca(2+)-induced Ca(2+)-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. CONCLUSION: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Patient with Kabuki syndrome and acute leukemia.

Kabuki syndrome is a multiple congenital anomaly/mental retardation syndrome which often involves recurrent infections. There is cumulative evidence of an immunodeficiency in Kabuki patients. We report a 2-year-old girl with typical Kabuki syndrome, who developed acute lymphocytic leukemia. The patient showed low levels of immunoglobulins G and A and a history of recurrent infections, that might indicate an immunodeficiency leading to an increased susceptibility to cancer. The girl was treated according to BFM protocols adapted to the patient's impaired cardiac situation and severe underweight. She achieved continual complete remission. Classical and molecular cytogenetic analyzes did not detect any abnormality.