Mitochondrial DNA variants in a cohort from Argentina with suspected Leber's hereditary optic neuropathy (LHON).

The present study investigates the spectrum and analysis of mitochondrial DNA (mtDNA) variants associated with Leber hereditary optic neuropathy (LHON) in an Argentinean cohort, analyzing 3 LHON-associated mitochondrial genes. In 32% of the cases, molecular confirmation of the diagnosis could be established, due to the identification of disease-causing variants. A total of 54 variants were observed in a cohort of 100 patients tested with direct sequencing analysis. The frequent causative mutations m.11778G>A in MT-ND4, m.3460G>A in MT-ND1, and m.14484T>C in MT-ND6 were identified in 28% of the cases of our cohort. Secondary mutations in this Argentinean LHON cohort were m.11253T>C p.Ile165Thr in MT-ND4, identified in three patients (3/100, 3%) and m.3395A>G p.Tyr30Cys in MT-ND1, in one of the patients studied (1%). This study shows, for the first time, the analysis of mtDNA variants in patients with a probable diagnosis of LHON in Argentina. Standard molecular methods are an effective first approach in order to achieve genetic diagnosis of the disease, leaving NGS tests for those patients with negative results.

Posterior Amorphous Corneal Dystrophy: New Chromosomal Breakpoints in the Small Leucine-Rich Proteoglycan-Coding Region.

PURPOSE: The purpose of this study was to report the clinical features and describe the results obtained by multimodal corneal imaging of a patient with novel chromosomal breakpoints of the 12q21.33 locus. METHODS: This study was a case report and literature review. RESULTS: A 12-year-old girl presented with visual loss whose examination revealed a best-corrected visual acuity of 20/50 in her right eye and 20/35 in her left eye and corneal flattening and gray sheet-like opacities deep in the stroma. Anterior segment optical coherence tomography and ultrabiomicroscopy showed an evenly distributed hyperreflective line in the posterior stroma. Confocal microscopy revealed enlarged keratocytes and the presence of small reflective deposits from the pre-Descemet line to the endothelium. In addition, a 447-kb deletion that included the small leucine-rich proteoglycan-coding region in locus 12q21.33 was found. She was, therefore, diagnosed with PACD. CONCLUSIONS: PACD is a rare genetic disorder of the cornea characterized by gray sheet-like opacification of the posterior stroma in combination with corneal flattening. Confocal microscopy provides histologic segmentation of each corneal layer and shows the degree to which they are affected. New chromosomal breakpoints of a deletion in the small leucine-rich proteoglycan-coding region are hereby reported. PACD may be a contiguous gene syndrome, and further tests are required to identify the exact position responsible for the phenotypic variation.

Predicting pathogenicity for novel hearing loss mutations based on genetic and protein structure approaches.

Hearing loss is a heterogeneous disorder. Identification of causative mutations is demanding due to genetic heterogeneity. In this study, we investigated the genetic cause of sensorineural hearing loss in patients with severe/profound deafness. After the exclusion of GJB2-GJB6 mutations, we performed whole exome sequencing in 32 unrelated Argentinean families. Mutations were detected in 16 known deafness genes in 20 patients: ACTG1, ADGRV1 (GPR98), CDH23, COL4A3, COL4A5, DFNA5 (GSDDE), EYA4, LARS2, LOXHD1, MITF, MYO6, MYO7A, TECTA, TMPRSS3, USH2A and WSF1. Notably, 11 variants affecting 9 different non-GJB2 genes resulted novel: c.12829C > T, p.(Arg4277*) in ADGRV1; c.337del, p.(Asp109*) and c.3352del, p.(Gly1118Alafs*7) in CDH23; c.3500G > A, p.(Gly1167Glu) in COL4A3; c.1183C > T, p.(Pro395Ser) and c.1759C > T, p.(Pro587Ser) in COL4A5; c.580 + 2 T > C in EYA4; c.1481dup, p.(Leu495Profs*31) in LARS2; c.1939 T > C, p.(Phe647Leu), in MYO6; c.733C > T, p.(Gln245*) in MYO7A and c.242C > G, p.(Ser81*) in TMPRSS3 genes. To predict the effect of these variants, novel protein modeling and protein stability analysis were employed. These results highlight the value of whole exome sequencing to identify candidate variants, as well as bioinformatic strategies to infer their pathogenicity.

Analysis of complex structural variants in the DMD gene in one family.

This work describes a family with Duchenne muscular dystrophy (DMD) with a rare case of a symptomatic pregnant woman. The main aim was to perform prenatal molecular diagnosis to provide genetic counseling. The secondary aim was to suggest the molecular mechanisms causing the complex structural variant (cxSV) identified. To accomplish this, we used a multi-technique algorithm including segregation analysis, Multiplex Ligation-dependent Probe Amplification, PCR, X-chromosome inactivation studies, microarrays, whole genome sequencing and bioinformatics. We identified a duplication of exons 38-43 in the DMD gene in all affected and obligate carrier members, proving that this was the DMD-causing mutation. We also observed a skewed X-chromosome inactivation in the symptomatic woman that explained her symptomatology. In addition, we identified a cxSV (duplication of exons 38-43 and deletion of exons 45-54) in the affected boy. The molecular characterization and bioinformatic analyses of the breakpoint junctions allowed us to identify Double Strand Breaks stimulator motifs and suggested the replication-dependent Fork Stalling and Template Switching as the most probable mechanisms leading to the duplication. In addition, the de novo deletion might have been the result of a germline inter-chromosome non-allelic recombination involving the Non-Homologous End Joining mechanism. In conclusion, the diagnostic strategy used allowed us to provide accurate molecular diagnosis and genetic counseling. In addition, the familial molecular diagnosis together with the in-depth characterization of the cxSV helped to determine the chronology of the molecular events, and propose and understand the molecular mechanisms involved in the generation of this complex rearrangement.

Multiple copy number variants in a pediatric patient with Hb H disease and intellectual disability.

Two distinct syndromes that link α-thalassemia and intellectual disability (ID) have been described: ATR-X, due to mutations in the ATRX gene, and ATR-16, a contiguous gene deletion syndrome in the telomeric region of the short arm of chromosome 16. A critical region where the candidate genes for the ID map has been established. In a pediatric patient with Hemoglobin H disease, dysmorphic features and ID, 4 novel and clinically relevant Copy Number Variants were identified. PCR-GAP, MLPA and FISH analyses established the cause of the α-thalassemia. SNP-array analysis revealed the presence of 4 altered loci: 3 deletions (arr[hg19]Chr16(16p13.3; 88,165-1,507,988) x1; arr[hg19]Chr6(6p21.1; 44,798,701-45,334,537) x1 and arr[hg19]Chr17(17q25.3; 80,544,855-81,057,996) x1) and a terminal duplication (arr[hg19]Chr7(7p22.3-p22.2; 4,935-4,139,785) x3). The -α(3.7) mutation and the ∼1.51 Mb in 16p13.3 are involved in the alpha-thalassemic phenotype. However, the critical region for ATR-16 cannot be narrowed down. The deletion affecting 6p21.1 removes the first 2 exons and part of intron 2 of the RUNX2 gene. Although heterozygous loss of function mutations affecting this gene have been associated with cleidocranial dysplasia, the patient does not exhibit pathognomonic signs of this syndrome, possibly due to the fact that the isoform d of the transcription factor remains unaffected. This work highlights the importance of searching for cryptic deletions in patients with ID and reiterates the need of the molecular analysis when it is associated to microcytic hypochromic anemia with normal iron status.

[Molecular bases of α-thalassemia in Argentina]. / Bases moleculares de alfa-talasemia en la Argentina.

The α-thalassemia is one of the most common hereditary disorders worldwide. Currently, molecular diagnostics is the only available tool to achieve an accurate diagnosis. The purpose of this study was to characterize the molecular bases of these syndromes in our environment and to establish genotype-phenotype associations. Through a combination of different molecular techniques and fluorescent in situ hybridization (FISH),we were able to find α-thalassemic mutations in 145 of the 184 patients (78.8%) studied with hematological parameters compatible with α-thalassemia. Deletions of the α-globin genes resulted the major molecular cause of the disease, and the most frequent mutation was -α(3.7), found in homozygous and heterozygous genotypes. In patients with α° phenotypes, other prevalent mutations were( _MED) and (_CAL/CAMP). The description of a sub-telomeric deletion in a patient with α-thalassemia and mental retardation was also achieved. ß-thalassemic mutations in heterozygous state were found in 7.6% of the patients, who presented α-thalassemic clinical features (microcytosis and Hb A2levels below 3.5%). Hematologic profiles for the α+ and α° genotypes were established for adult and pediatric patients. Hopefully, this work will provide guidelines for the detection of possible α-thalassemic carriers. It also highlights the collaborative work of hematologists, the biochemical and molecular biology laboratory and genetists, in order to provide appropriate genetic counseling.

Bases moleculares de alfa-talasemia en la Argentina

La α-talasemia, es uno de los desórdenes hereditarios más frecuentes mundialmente. Al presente, el diagnóstico molecular es la única herramienta que permite el diagnóstico certero. El propósito de este trabajo fue caracterizar las bases moleculares de estos síndromes en nuestro medio, y establecer relaciones genotipo-fenotipo. Mediante la complementación de distintas técnicas de biología molecular e hibridación fluorescente in situ (FISH), se logró poner en evidencia la presencia de mutaciones α-talasémicas en 145 de 184 (78.8%) pacientes estudiados con perfil hematológico compatible con α-talasemia. Dentro de este grupo, las deleciones correspondieron al defecto genético más frecuente, prevaleciendo la mutación -α3.7 en genotipos heterocigotas y homocigotas. Asimismo, en pacientes con fenotipo α0 las deleciones prevalentes fueron -MED y -CAL/CAMP. Este estudio permitió también describir una deleción de la región sub-telomérica en un paciente con α-talasemia y retraso mental. En el 7.6% de los pacientes caracterizados clínicamente como posibles α-talasémicos (microcitosis con valores de Hb A2 inferiores al 3.5%), se hallaron mutaciones β-talasémicas en estado heterocigota. Se lograron establecer perfiles hematológicos asociados a los genotipos α+ y α0 para pacientes adultos y niños. Esperamos que este trabajo pueda servir como guía para reconocer posibles portadores α-talasémicos. También permite destacar el trabajo en conjunto de médicos hematólogos, el laboratorio (bioquímico y de biología molecular) y de los médicos genetistas, con el fin de proporcionar adecuado consejo genético.

The α-thalassemia is one of the most common hereditary disorders worldwide. Currently, molecular diagnostics is the only available tool to achieve an accurate diagnosis. The purpose of this study was to characterize the molecular bases of these syndromes in our environment and to establish genotype-phenotype associations. Through a combination of different molecular techniques and fluorescent in situ hybridization (FISH),we were able to find α-thalassemic mutations in 145 of the 184 patients (78.8%) studied with hematological parameters compatible with α-thalassemia. Deletions of the a-globin genes resulted the major molecular cause of the disease, and the most frequent mutation was -α3.7, found in homozygous and heterozygous genotypes. In patients with α0 phenotypes, other prevalent mutations were -MED and -CAL/CAMP. The description of a sub-telomeric deletion in a patient with α-thalassemia and mental retardation was also achieved. β-thalassemic mutations in heterozygous state were found in 7.6% of the patients, who presented α-thalassemic clinical features (microcytosis and Hb A2 levels below 3.5%). Hematologic profiles for the α+ and α0 genotypes were established for adult and pediatric patients. Hopefully, this work will provide guidelines for the detection of possible α-thalassemic carriers. It also highlights the collaborative work of hematologists, the biochemical and molecular biology laboratory and genetists, in order to provide appropriate genetic counseling.

Bases moleculares de alfa-talasemia en la Argentina

La α-talasemia, es uno de los desórdenes hereditarios más frecuentes mundialmente. Al presente, el diagnóstico molecular es la única herramienta que permite el diagnóstico certero. El propósito de este trabajo fue caracterizar las bases moleculares de estos síndromes en nuestro medio, y establecer relaciones genotipo-fenotipo. Mediante la complementación de distintas técnicas de biología molecular e hibridación fluorescente in situ (FISH), se logró poner en evidencia la presencia de mutaciones α-talasémicas en 145 de 184 (78.8%) pacientes estudiados con perfil hematológico compatible con α-talasemia. Dentro de este grupo, las deleciones correspondieron al defecto genético más frecuente, prevaleciendo la mutación -α3.7 en genotipos heterocigotas y homocigotas. Asimismo, en pacientes con fenotipo α0 las deleciones prevalentes fueron -MED y -CAL/CAMP. Este estudio permitió también describir una deleción de la región sub-telomérica en un paciente con α-talasemia y retraso mental. En el 7.6% de los pacientes caracterizados clínicamente como posibles α-talasémicos (microcitosis con valores de Hb A2 inferiores al 3.5%), se hallaron mutaciones β-talasémicas en estado heterocigota. Se lograron establecer perfiles hematológicos asociados a los genotipos α+ y α0 para pacientes adultos y niños. Esperamos que este trabajo pueda servir como guía para reconocer posibles portadores α-talasémicos. También permite destacar el trabajo en conjunto de médicos hematólogos, el laboratorio (bioquímico y de biología molecular) y de los médicos genetistas, con el fin de proporcionar adecuado consejo genético.(AU)

The α-thalassemia is one of the most common hereditary disorders worldwide. Currently, molecular diagnostics is the only available tool to achieve an accurate diagnosis. The purpose of this study was to characterize the molecular bases of these syndromes in our environment and to establish genotype-phenotype associations. Through a combination of different molecular techniques and fluorescent in situ hybridization (FISH),we were able to find α-thalassemic mutations in 145 of the 184 patients (78.8%) studied with hematological parameters compatible with α-thalassemia. Deletions of the a-globin genes resulted the major molecular cause of the disease, and the most frequent mutation was -α3.7, found in homozygous and heterozygous genotypes. In patients with α0 phenotypes, other prevalent mutations were -MED and -CAL/CAMP. The description of a sub-telomeric deletion in a patient with α-thalassemia and mental retardation was also achieved. β-thalassemic mutations in heterozygous state were found in 7.6% of the patients, who presented α-thalassemic clinical features (microcytosis and Hb A2 levels below 3.5%). Hematologic profiles for the α+ and α0 genotypes were established for adult and pediatric patients. Hopefully, this work will provide guidelines for the detection of possible α-thalassemic carriers. It also highlights the collaborative work of hematologists, the biochemical and molecular biology laboratory and genetists, in order to provide appropriate genetic counseling.(AU)

Prenatal diagnosis of Duchenne/Becker muscular dystrophy by short tandem repeat segregation analysis in Argentine families.

INTRODUCTION: Duchenne/Becker muscular dystrophies (DMD/BMD) are X-linked recessive diseases caused by mutations in the dystrophin gene. METHODS: We used multiplex polymerase chain reaction (PCR) and short tandem repeat (STR) segregation analysis for DMD/BMD-carrier detection and prenatal diagnosis. RESULTS: Twenty-four at-risk pregnancies were evaluated: 17 were excluded from carrying dystrophin gene mutations with 95-100% certainty. Of the remaining cases, 2 were determined to carry a dystrophin gene mutation with 95-100% certainty. Three cases had a 67% probability of carrying the mutation, and 2 others were not informative. The certainty of the test increased to ~100% in some cases due to the identification of several genetic events: 4 recombinations; 4 de novo mutations; and 8 deletions encompassing some of the STRs evaluated. DISCUSSION: Overall, 19 of 24 (79%) molecular prenatal diagnoses were informative, indicating that multiplex PCR/STR segregation analysis is a reliable method for carrier detection and prenatal diagnosis when other more sophisticated techniques are unavailable.

GJB2 and GJB6 genes: molecular study and identification of novel GJB2 mutations in the hearing-impaired Argentinean population.

Mutations in the GJB2 gene are responsible for more than half of all cases of recessive non-syndromic deafness. This article presents a mutation analysis of the GJB2, GJB6, OTOF and MTRNR1 genes in 252 patients with sensorineural non-syndromic hearing loss. Thirty-one different mutations were identified in GJB2 and GJB6 in 86 of the 252 (34%) patients. We describe for the first time two new mutations in GJB2: the missense mutation c.29 T>C (p.Leu10Pro) in the N terminal domain and c.326 G>T (p.Gly109Val) in the intracytoplasmic domain of connexin 26. This work shows the high prevalence of GJB2 mutations in the Argentinean population, with frequencies that are comparable to those of the Mediterranean area. Most important, it adds two novel GJB2 mutations to be taken into consideration in the genetic diagnosis of non-syndromic sensorineural hearing loss.

Asymptomatic Becker muscular dystrophy in a family with a multiexon deletion.

We report a Becker muscular dystrophy (BMD) family with one 5-year-old affected patient and a 69-year-old asymptomatic grandfather. Dystrophin gene multiplex polymerase chain reaction and multiplex ligation-dependant probe amplification analysis showed that both males carried an in-frame deletion of exons 45-55. Segregation analysis revealed two additional asymptomatic boys in this family. Our finding supports previous predictions that exons 45-55 are the optimal multiexon skipping target in antisense gene therapy to transform the severe Duchenne muscular dystrophy into the milder BMD, or even asymptomatic, phenotype.

The role of polymorphic short tandem (CA)n repeat loci segregation analysis in the detection of Duchenne muscular dystrophy carriers and prenatal diagnosis.

BACKGROUND: Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked diseases caused by mutations in the dystrophin gene at Xp21.2; they include gross deletions (60%), duplications (10%), and small mutations (30%). Since there is no cure or effective treatment for progressive muscular dystrophy, prevention of the disease is important and strongly depends on carrier-status information. Two-thirds of DMD/BMD cases are familial; thus, female relatives are candidates for carrier-risk assessment. AIM: Segregation analysis of polymorphic short tandem (CA)n repeats [STR-(CA)n] was used to establish and compare the haplotypes of female relatives of patients with DMD/BMD with those of the patient in order to identify the mutant dystrophin gene and thus determine each female relative's carrier status. METHODS: 248 individuals from 52 families were studied through segregation of up to 11 STR-(CA)n loci. The assay was performed on leukocyte DNA by PCR amplification, polyacrylamide-gel electrophoresis and autoradiography. Haplotypes were established by determination of alleles on the autoradiography. RESULTS: 38 of 51 (75%) female relatives from familial cases were diagnosed as carriers or non-carriers with a 95-100% likelihood, and 18 out of 56 (32%) female relatives from sporadic cases could be excluded from the risk of being a DMD carrier with the same probability. In addition, STR studies detected gross deletions in 13 of the 52 (25%) families in both male and female individuals, four of which were de novo deletions. STR assays were also informative in families without an available DNA sample of an affected male and in two of seven symptomatic females. Determination of carrier status was particularly significant for prediction of DMD risk in prenatal analysis of five male chorionic villi. Other genetic events revealed by STR analysis were: (i) 11 recombinations identified in 6.6% of meiosis in the DMD families; (ii) germinal mosaicism detected in two female carriers; and (iii) changes in STR-(CA)n length during transmission from father to daughters, including three retractions and one elongation at an estimated rate of 0.004. CONCLUSION: The STR assay is an excellent molecular tool for carrier-status identification and the detection of deletions and other genetic changes in families affected by DMD/BMD. Thus, it is useful in genetic counseling for the prevention of this disease.

Combined cytogenetic and molecular analyses for the diagnosis of Prader-Willi/Angelman syndromes.

Prader-Willi (PWS) and Angelman (AS) are syndromes of developmental impairment that result from the loss of expression of imprinted genes in the paternal (PWS) or maternal (AS) 15q11-q13 chromosome. Diagnosis on a clinical basis is difficult in newborns and young infants; thus, a suitable molecular test capable of revealing chromosomal abnormalities is required. We used a variety of cytogenetic and molecular approaches, such as, chromosome G banding, fluorescent in situ hybridization, a DNA methylation test, and a set of chromosome 15 DNA polymorphisms to characterize a cohort of 27 PWS patients and 24 suspected AS patients. Molecular analysis enabled the reliable diagnosis of 14 PWS and 7 AS patients, and their classification into four groups: (A) 6 of these 14 PWS subjects (44 %) had deletions of paternal 15q11-q13; (B) 4 of the 7 AS patients had deletions of maternal 15q11-q13; (C) one PWS patient (8 %) had a maternal uniparental disomy (UPD) of chromosome 15; (D) the remaining reliably diagnoses of 7 PWS and 3 AS cases showed abnormal methylation patterns of 15q11-q13 chromosome, but none of the alterations shown by the above groups, although they may have harbored deletions undetected by the markers used. This study highlights the importance of using a combination of cytogenetic and molecular tests for a reliable diagnosis of PWS or AS, and for the identification of genetic alterations.