Viviana Dalamon

RB1 Germ-Line Deletions in Argentine Retinoblastoma Patients

AbstractBackground: Retinoblastoma (RB) is a malignant tumor originating in the retinal cell precursors and can be presented as a unilateral or bilateral form in childhood (one or both eyes affected). Development of this tumor is caused by mutations in the RB1 gene on chromosome 13ql4; the first mutation may occur in the germ line (hereditary RB) or in somatic cells (non-hereditary RB). The hereditary form of RB is transmitted with a high penetrance to offspring (90%). Because early diagnosis is necessary for implementing effective treatment and preserving vision, it is important to identify the mutations in the affected family. Aim: The aim of this study was to identify large and small RB1 germ-line mutations and to correlate them with the RB phenotype. Methods: Constitutional RB1 gene gross deletions were studied in 40 patients with bilateral or unilateral familial RB by a segregation assay of four intragenic polymorphisms located in introns 1,4, 17, and 20 of the RB1 gene, along with fluorescence in situ hibridization (FISH) analysis. Small mutations were ascertained in a subgroup of ten patients by heteroduplex/sequence analysis of RB1-exons. Results: In the course of our study, we have found three large deletions, which probably represent whole gene deletions, and two small deletions of 1bp in length. One large deletion was found in a family with several members affected. This represents a rare case of familial RB, which is usually caused by small mutations. Phenotype analysis of the family revealed a low penetrance inheritance, with an ‘affected eyes: number of mutation-carriers’ ratio of ≈1.0, whereas this ratio in families with small loss-of-function mutations is 1.5–2.0. Conclusions: Our results emphasize the usefulness of a combined methodology that includes segregation of polymorphisms, FISH, and heteroduplex/sequence analyses for detection of gross and small DNA rearrangements in familial and sporadic RB. Identification of mutations in sporadic cases is important for risk-assessment in patients’ relatives. The degree of penetrance in the inheritance of RB not only depends on the occurrence of the second mutation in the RB1 gene but also on the extent of inactivation of the first mutation.

Electrical Properties and Functional Expression of Ionic Channels in Cochlear Inner Hair Cells of Mice Lacking the α10 Nicotinic Cholinergic Receptor Subunit

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, synaptic transmission is triggered by spontaneous Ca2+ spikes which are modulated by an efferent cholinergic innervation to IHCs. This synapse is inhibitory and mediated by the α9α10 nicotinic cholinergic receptor (nAChR). After the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired and both the spiking activity and the efferent innervation disappear from IHCs. In this work, we studied the developmental changes in the membrane properties of cochlear IHCs from α10 nAChR gene (Chrna10) “knockout” mice. Electrophysiological properties of IHCs were studied by whole-cell recordings in acutely excised apical turns of the organ of Corti from developing mice. Neither the spiking activity nor the developmental functional expression of voltage-gated and/or calcium-sensitive K+ channels is altered in the absence of the α10 nAChR subunit. The present results show that the α10 nAChR subunit is not essential for the correct establishment of the intrinsic electrical properties of IHCs during development.

Carrier detection in Duchenne and Becker muscular dystrophy Argentine families

In order to offer carrier detection, genetic counseling, and prenatal diagnosis to families with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) in our country, segregation analysis of highly polymorphic short tandem repeats (STR) (dC‐dA)n: (dG‐dT)n loci was utilized. The risks to females of 15 DMD/BMD families (9 familial and 6 sporadic) were evaluated on STR, pedigree and serum creatine kinase (SCK) data. From the 36 females at risk of being carriers (not including 8 obligate carriers), results of STR analysis were compatible with carrier status in 7 and not compatible in 20. In 9 females, no information regarding carriership was derived from the STR analysis. Prenatal diagnosis is now possible on the carrier females. Previously identified deletions in the central part of the gene were confirmed by STR analysis in 3 families. Five new alleles were identified in Argentine individuals; allele frequencies differed from those of North American people. Results derived from this study are useful for carrier detection and genetic counseling in DMD/BMD. One case of probable mosaicism in an unaffected father was detected on a pedigree basis in a family with DMD patients.

MLPA analysis of an Argentine cohort of patients with dystrophinopathy: Association of intron breakpoints hot spots with STR abundance in DMD gene.

Dystrophinopathies are X-linked recessive diseases caused by mutations in the DMD gene. Our objective was to identify mutations in this gene by Multiplex Ligation Probe Amplification (MLPA), to confirm the clinical diagnosis and determine the carrier status of at-risk relatives. Also, we aimed to characterize the Dystrophinopathies argentine population and the DMD gene. We analyzed a cohort of 121 individuals (70 affected boys, 11 symptomatic women, 37 at-risk women and 3 male villus samples). The MLPA technique identified 56 mutations (45 deletions, 9 duplications and 2 point mutations). These results allowed confirming the clinical diagnosis in 63% (51/81) of patients and symptomatic females. We established the carrier status of 54% (20/37) of females at-risk and 3 male villus samples. We could establish an association between the most frequent deletion intron breakpoints and the abundance of dinucleotide microsatellites loci, despite the underlying mutational molecular mechanism remains to be elucidated. The MLPA demonstrate, again, to be the appropriate first mutation screening methodology for molecular diagnosis of Dystrophinopathies. The reported results permitted to characterize the Dystrophinopathies argentine population and lead to better understanding of the genetic and molecular basis of rearrangements in the DMD gene, useful information for the gene therapies being developed.