Vera Beyer

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 Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-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.

Disruption of CNTNAP2 and additional structural genome changes in a boy with speech delay and autism spectrum disorder

Patients with autism spectrum disorder (ASD) frequently harbour chromosome rearrangements and segmental aneuploidies, which allow us to identify candidate genes. In a boy with mild facial dysmorphisms, speech delay and ASD, we reconstructed by karyotyping, FISH and SNP array-based segmental aneuploidy profiling a highly complex chromosomal rearrangement involving at least three breaks in chromosome 1 and seven breaks in chromosome 7. Chromosome banding revealed an inversion of region 7q32.1–7q35 on the derivative chromosome 7. FISH with region-specific BACs mapped both inversion breakpoints and revealed additional breaks and structural changes in the CNTNAP2 gene. Two gene segments were transposed and inserted into the 1q31.2 region, while the CNTNAP2 segment between the two transposed parts as well as intron 13 to the 5-UTR were retained on the der(7). SNP array analysis revealed an additional de novo deletion encompassing the distal part of intron1 and exon 2 of CNTNAP2, which contains FOXP2 binding sites. Second, we found another de novo deletion on chromosome 1q41, containing 15 annotated genes, including KCTD3 and USH2A. Disruptions of the CNTNAP2 gene have been associated with ASD and with Gilles de la Tourette syndrome (GTS). Comparison of disruptions of CNTNAP2 in patients with GTS and ASD suggests that large proximal disruptions result in either GTS or ASD, while relatively small distal disruptions may be phenotypically neutral. For full-blown ASD to develop, a proximal disruption of CNTNAP2 may have to occur concomitantly with additional genome mutations such as hemizygous deletions of the KCTD3 and USH2A genes.

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.

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 patients impaired cardiac situation and severe underweight. She achieved continual complete remission. Classical and molecular cytogenetic analyzes did not detect any abnormality.

Three de novo losses and one insertion within a pericentric inversion of chromosome 6 in a patient with complete absence of expressive speech and reduced pain perception

A 32-year-old female patient, observed for 30 years because of a distinctive phenotype consisting of a dysmorphic face non-progressive deficit of motor control, lack of speech development, reduced sensitivity to pain, with a known, complex interstitial deletion 6q14 within a de novo pericentric inversion 6p11.2;q15, was re-examined at the molecular level. Applying the Infinium HumanHap300 BeadChip array and BAC-based FISH we found two new non-contiguous microdeletions in addition to the one detected previously by high resolution G-band analysis. A 360 kb loss in band 6p12.3, containing the genes RHAG, CRISP1, 2, and 3, and PGK2, a 1.15 Mb loss in 6p12.2-p12.1, containing the genes PKHD1, IL17, MCM3, EFHC1, and TRAM2 genes, and an 11.9 Mb loss in region 6q14.3-q16.1, reported previously, were mapped on the rearranged chromosome 6. The latter loss contained the central cannabinoid receptor isoform b (CNR1), which may be involved in brain development and function. Since the maternal SNPs were retained this rearrangement of chromosome 6 is most likely of paternal origin.

Fulminant hepatic failure requiring liver transplantation in 22q13.3 deletion syndrome.

We report on a 4‐year‐old girl with severe developmental delay, absent speech, and chromosome 22q13.3 deletion (Phelan–McDermid syndrome), karyotype 46,XX.ish del(22)(q13.31qter)(ARSA‐,N85A‐,SHANK3‐). At the age of 3 years, she needed an emergency liver transplantation because of fulminant hepatic failure, most likely caused by hyperacute autoimmune hepatitis triggered by a viral infection. This is the second report of a patient with 22q13.3 deletion and fulminant liver failure. By array‐CGH we identified in this patient a 5.675 Mb terminal deletion (22q13.31 → qter; including ∼55 genes; from NUP50 to RABL2B) and in the previous patient a 1.535 Mb deletion (22q13.32 → qter; including ∼39 genes; from BRD1 to RABL2B). PIM3 is a prime candidate gene for the fulminant hepatic failure in the two patients; SHANK3/PROSAP2 could be another candidate gene. We recommend liver function tests and array‐CGH in the management of patients with Phelan–McDermid syndrome. This patient showed a developmental catch‐up following the liver transplantation, possibly suggesting that chronic hepatic disease could contribute to the developmental delay in a subset of these patients.

Two independent chromosomal rearrangements, a very small (550 kb) duplication of the 7q subtelomeric region and an atypical 17q11.2 (NF1) microdeletion, in a girl with neurofibromatosis

Most patients with neurofibromatosis (NF1) are endowed with heterozygous mutations in the NF1 gene. Approximately 5% show an interstitial deletion of chromosome 17q11.2 (including NF1) and in most cases also a more severe phenotype. Here we report on a 7-year-old girl with classical NF1 signs, and in addition mild overgrowth (97th percentile), relatively low OFC (10th–25th percentile), facial dysmorphy, hoarse voice, and developmental delay. FISH analysis revealed a 17q11.2 microdeletion as well as an unbalanced 7p;13q translocation leading to trisomy of the 7q36.3 subtelomeric region. The patient’s mother and grandmother who were phenotypically normal carried the same unbalanced translocation. The 17q11.2 microdeletion had arisen de novo. Array comparative genomic hybridization (CGH) demonstrated gain of a 550-kb segment from 7qter and loss of 2.5 Mb from 17q11.2 (an atypical NF1 microdeletion). We conclude that the patient’s phenotype is caused by the atypical NF1 deletion, whereas 7q36.3 trisomy represents a subtelomeric copy number variation without phenotypic consequences. To our knowledge this is the first report that a duplication of the subtelomeric region of chromosome 7q containing functional genes (FAM62B, WDR60, and VIPR2) can be tolerated without phenotypic consequences. The 17q11.2 microdeletion (containing nine more genes than the common NF1 microdeletions) and the 7qter duplication were not accompanied by unexpected clinical features. Most likely the 7qter trisomy and the 17q11.2 microdeletion coincide by chance in our patient.

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.

Disruption of the ATE1 and SLC12A1 Genes by Balanced Translocation in a Boy with Non-Syndromic Hearing Loss.

We report on a boy with non-syndromic hearing loss and an apparently balanced translocation t(10;15)(q26.13;q21.1). The same translocation was found in the normally hearing brother, father and paternal grandfather; however, this does not exclude its involvement in disease pathogenesis, for example, by unmasking a second mutation. Breakpoint analysis via FISH with BAC clones and long-range PCR products revealed a disruption of the arginyltransferase 1 (ATE1) gene on translocation chromosome 10 and the solute carrier family 12, member 1 gene (SLC12A1) on translocation chromosome 15. SNP array analysis revealed neither loss nor gain of chromosomal regions in the affected child, and a targeted gene enrichment panel consisting of 130 known deafness genes was negative for pathogenic mutations. The expression patterns in zebrafish and humans did not provide evidence for ear-specific functions of the ATE1 and SLC12A1 genes. Sanger sequencing of the 2 genes in the boy and 180 GJB2 mutation-negative hearing-impaired individuals did not detect homozygous or compound heterozygous pathogenic mutations. Our study demonstrates the many difficulties in unraveling the molecular causes of a heterogeneous phenotype. We cannot directly implicate disruption of ATE1 and/or SLC12A1 to the abnormal hearing phenotype; however, mutations in these genes may have a role in polygenic or multifactorial forms of hearing impairment. On the other hand, it is conceivable that our patient carries a disease-causing mutation in a so far unidentified deafness gene. Evidently, disruption of ATE1 and/or SLC12A1 gene function alone does not have adverse effects.

Haploinsufficiency of 16.4 Mb from Chromosome 22pter-q11.21 in a Girl with Unilateral Conductive Hearing Loss

We present the postnatal diagnosis of a de novo der(18)t(18;22)(p11.32;q11.21)pat, resulting in an unbalanced 45,XX,der (18)t(18;22) karyotype in a girl with conductive hearing loss on the left and ptosis of the right upper eye-lid. Unilateral ptosis was also observed in the patient’s 2 years and 8 months younger sister, who grows noticeably faster and appears to be a much quicker learner. After speech therapy the patient was eventually placed in normal school. The haploinsufficient 16.4-Mb region on chromosome 22pter→q11.21 contains 10 genes as well as many predicted genes, pseudogenes, and retrotransposed sequences with unknown functions. This observation may prove useful for prenatal diagnosis and genetic counselling of chromosome 22q11.1 gains and losses.