Luciana A. Haddad

A PCR-based test suitable for screening for fragile X syndrome among mentally retarded males

Ever since the identification of the genetic cause of fragile X syndrome as the expansion of an unstable trinucleotide sequence, several diagnostic strategies have evolved from molecular studies. However, we still lack a simple test suitable for population screening. We have therefore developed a nonisotopic polymerase chain reaction (PCR)-based technique for the identification of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacrylamide gels. The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 by for the (CGG)29 allele. Conditions were established such that full mutations failed to amplify and were thus identified with 98% sensitivity compared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 by upstream of the polymorphic region. In trials involving 41 patients and 74 controls, the PCR-based test here described showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. Thus, although not suitable for medical diagnosis, it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males.

Fully mutated and gray-zone FRAXA alleles in Brazilian mentally retarded boys

We used a non-isotopic polymerase chain reaction (PCR) technique for fragile X syndrome diagnosis to screen 256 mentally retarded boys who were selected randomly from special schools. Patients identified as pre- or full-mutation carriers were further investigated by Southern blot analysis with the StB12.3 probe. The PCR-based test identified five boys with the expanded allele and 17 other patients as carriers of either premutated or gray-zone alleles. The full mutation was confirmed in four cases after Southern blotting and a fifth patient carried a normal allele. Of the 17 patients identified with a premutation allele by PCR, one individual was diagnosed as mosaic by Southern blotting, 12 individuals displayed fragments of 2.90 kb or 2.85 kb, and the remaining four individuals showed apparently normal-sized fragments. However, sizing of these 16 alleles by further PCR analysis showed them to be in the gray-zone range (40-60 repeats). Therefore, the frequency of the full mutation in this cohort of mentally retarded boys was close to 2% (5/256). The prevalence of gray-zone alleles among those mentally impaired boys who did not carry the full mutation was 6.4% (16/251) and, although more than twice the prevalence of these alleles among a cohort of unaffected Brazilian males 2.8% (71251), the difference did not reach statistical significance.

The number of CGG repeats of the FMR1 locus in premutated and fully mutated heterozygotes and their offspring: Implications for the origin of mosaicism

The size of the CGG repeat of the FMR1 gene was investigated with probe StB12.3 in 154 transmissions to the offspring of heterozygotes for the premutation and the full mutation. Among the 135 offspring of premutated heterozygotes there were three decreases in size of the repeats: in two of these cases a full mutation was present along with the decreased premutation, and in a third mosaic (46,fra(X)(q27.3),Y), a normal allele was observed. In the 19 offspring of fully mutated females with no detected mosaicism, there were three mosaics and three individuals who had full mutations that included a number of repeats smaller than those present in their mothers. Among the 32 offspring who received a premutation from their premutated mothers, 27 alleles were increased in size and 5 remained unaltered. Among 11 mosaic offspring of premutated mothers, the premutation increased in 4, decreased in 3, and was unchanged in 4. In contrast to the trend of an increasing premutation size in the non-mosaic offspring, the premutation present in mosaics can be smaller, larger, or of unaltered size with approximately equal frequencies. These data suggest that the premutations present in mosaics result from mitotic instability of the inherited full mutations. This is further supported by the finding of a mosaic male with a normal sized allele.

Simultaneous detection of size and sequence polymorphisms in the transcribed trinucleotide repeat D2S196E (EST00493)

Abstract Long expansions of transcribed trinucleotide microsatellites have been etiologically associated with some neurological diseases. The investigation of such novel polymorphisms has thus become a subject of great interest. We searched the expressed sequence tag databank for reiterated trinucleotides and selected EST00493 (D2S196E) with 14 tandem ACA triplets as a potentially polymorphic locus. Size variation was readily detected, with four common alleles containing 12–15 repeats. In addition, we observed distinct heteroduplexes in amplifications from individuals with identical ACA genotypes. Sequencing of their polymerase chain reaction (PCR) products revealed a G→A transition immediately preceding the trinucleotide repeats, hence defining 8 distinct haplotypes and 36 possible genotypes. Indeed, mutation detection enhancement gel electrophoresis of mixed PCR products from cloned haplotypes revealed 24 distinct heteroduplex patterns for the six possible trinucleotide heterozygotes. The observation of heteroduplex patterns in non-denaturing polyacrylamide gel electrophoresis (instead of the more commonly used denaturing gels) can thus be utilized to increase the informativeness of microsatellite polymorphisms by unraveling otherwise cryptic sequence variation. The D2S196E polymorphism has proved useful for demonstrating microsatellite instability and loss of heterozygosity in colorectal tumors.

Polarity of mutations in tumor-associated microsatellite instability

Abstract Many factors have been implicated in influencing the rate of microsatellite mutations, including the length and base composition of the repeat motif, number of repeats, base composition of flanking sequences and, perhaps most importantly, degree of perfection of the repeats. The latter is of clinical relevance, since in both spino-cerebellar ataxia and fragile X syndrome, alleles with imperfect repeats appear to be much more stable than perfect ones. As yet, the relative importance of increased replication slippage and decreased mismatch repair efficiency in the preference of mutations to occur within perfect repeats has not been fully determined. D13S308E is an asymmetric trinucleotide repeat microsatellite with the sequence (CAT)3CAC(CAT)CAC(CAT)2CAC(CAT)CAC(CAT)15, thus containing two parts: an 11-repeat imperfect portion (underlined above) and a 15-repeat perfect one (bold). We sequenced eight new mutant alleles of D13S308E from three human gastric tumors with instability in this and other microsatellites. In all mutations the size variation occurred exclusively in the perfect part of the microsatellite. These results constitute direct evidence that the molecular basis of microsatellite alterations seen in normal cells is similar to those that occur in human tumors with extensive microsatellite instability. The investigation of mechanisms involved in microsatellite mutations has been handicapped by the fact that they are rare events. The microsatellite instability observed in malignant tumors provides us with a useful general system to study these mechanisms.

A Cell Junctional Protein Network Associated with Connexin-26

GJB2 mutations are the leading cause of non-syndromic inherited hearing loss. GJB2 encodes connexin-26 (CX26), which is a connexin (CX) family protein expressed in cochlea, skin, liver, and brain, displaying short cytoplasmic N-termini and C-termini. We searched for CX26 C-terminus binding partners by affinity capture and identified 12 unique proteins associated with cell junctions or cytoskeleton (CGN, DAAM1, FLNB, GAPDH, HOMER2, MAP7, MAPRE2 (EB2), JUP, PTK2B, RAI14, TJP1, and VCL) by using mass spectrometry. We show that, similar to other CX family members, CX26 co-fractionates with TJP1, VCL, and EB2 (EB1 paralogue) as well as the membrane-associated protein ASS1. The adaptor protein CGN (cingulin) co-immuno-precipitates with CX26, ASS1, and TJP1. In addition, CGN co-immunoprecipitation with CX30, CX31, and CX43 indicates that CX association is independent on the CX C-terminus length or sequence. CX26, CGN, FLNB, and DAMM1 were shown to distribute to the organ of Corti and hepatocyte plasma membrane. In the mouse liver, CX26 and TJP1 co-localized at the plasma membrane. In conclusion, CX26 associates with components of other membrane junctions that integrate with the cytoskeleton.

Stem Cells within PGAt Observed In Vivo after 6 Weeks Enhance Facial Nerve Regeneration

Objectives: Autografting, the gold-standard method for facial nerve repair with tissue loss, in association with high quality scaffolds and cell implants, has disclosed distinct experimental outcomes. The aim of the study was to evaluate the functional and histological effects of bone marrow stem cells (BMSC) combined with polyglycolic acid tube (PGAt) in autografted rat facial nerves. Methods: After neurotmesis of the mandibular branch of the rat facial nerve, surgical repair consisted of nerve autografting (groups A-E), contained in PGAt (groups B-E), filled with basement membrane matrix (groups C-E), with undifferentiated BMSC (group D) or Schwann-like cells that had differentiated from BMSC (group E). Axon morphometrics and an objective compound muscle action potentials (CMAP) analysis were conducted. Immunofluorescence assays were carried out with Schwann cell marker S100 and anti-B-galactosidase to label exogenous cells. Results: Six weeks after surgery, animals from either cell-containing group had mean CMAP amplitudes significantly higher than control groups. Differently from other groups, facial nerves with Schwann-like cells implants had mean axonal densities within reference values. This same group had the highest mean axonal diameter in distal segments. We observed expression of the reporter gene LacZ in nerve cells in the graft and distally from it in groups D and E. Group E cells had LacZ coexpressed with S100. Conclusions: Regeneration of the facial nerve was improved by BMSC within PGAt in rats, yet Schwann-like cells were associated with superior effects. Accordingly, groups D and E had BMSC integrated in neural tissue with maintenance of former cell phenotype for 6 weeks.

Viable, Proliferating Progenitor Cells

Objective: Compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day 3 (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy. Method: Organs of Corti were isolated from P3 guinea pig or mouse cochlea, dissociated, and cultivated under nonadherent conditions as cell clusters (otospheres), in DMEM:F12 medium, supplemented with epidermal growth factor (EGF), plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFα), and submitted to immunofluorescence assays. Results: Otospheres from mouse and guinea pig organ of Corti cultivated in vitro retained properties of inner ear progenitor cells, such as self-renewal, proliferation, and differentiation into hair cells or supporting cells. The best culture outcome was observed when mouse-derived cells were cultivated in the presence of TGFα instead of bFGF. Otosphere cell sorting will be additionally tested by flow cytometry of dissociated mouse cells. These ongoing experiments will be useful for the phenotype characterization of the progenitor cells presenting the best proliferating index. Conclusion: The expression of sox2 and nestin in guinea pig and mouse otosphere is supporting evidence for the presence of inner ear progenitor cells in P3 guinea pig. However, the proliferation and differentiation potential of mouse-derived cells place this organism as a better model for studying early postnatal progenitor cell dynamics.