Autonomous B-cell receptor signaling and genetic aberrations in chronic lymphocytic leukemia-phenotype monoclonal B lymphocytosis in siblings of patients with chronic lymphocytic leukemia.

Clonal expansion of CD5-expressing B cells, commonly designated as monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL). The mechanisms driving subclinical MBL B-cell expansion and progression to CLL, occurring in approximately 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for the pathogenesis of CLL. The objectives of this study were functional characterization of the BCR of MBL in siblings of CLL patients and a comparison of genetic variants in MBL-CLL sibling pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per microliter (median: 37/µL) in 34 of 191 (17.8%) siblings of CLL patients. Clonal BCR isolated from highly purified CLL-phenotype cells induced robust calcium mobilization in BCR-deficient murine pre-B cells in the absence of external antigen and without experimental crosslinking. This autonomous BCR signal was less intense than the signal originating from the CLL BCR of their CLL siblings. According to genotyping by single nucleotide polymorphism array, whole exome, and targeted panel sequencing, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, respectively. Likewise, the prevalence of recurrent CLL-associated genetic variants was similar between CLL and matched MBL samples. However, copy number variations and small variants were frequently subclonal in MBL cells, suggesting their acquisition during subclinical clonal expansion. These findings support a stepwise model of CLL pathogenesis, in which autonomous BCR signaling leads to a non-malignant (oligo)clonal expansion of CD5+ B cells, followed by malignant progression to CLL after acquisition of pathogenic genetic variants.

An outbreak of SARS-CoV-2 in big hairy armadillos (Chaetophractus villosus) associated with Gamma variant in Argentina three months after being undetectable in humans

The present pandemic produced by SARS-CoV-2 and its variants represents an example of the one health concept in which humans and animals are components of the same epidemiologic chain. Animal reservoirs of these viruses are thus the focus of surveillance programs to monitor their circulation and evolution in potentially new hosts and reservoirs. In this work, we report the detection of SARS-CoV-2 Gamma variant infection in four specimens of Chaetophractus villosus (big hairy armadillo/armadillo peludo) in Argentina. In addition to the finding of a new wildlife species susceptible to SARS-CoV-2 infection, the identification of the Gamma variant three months after its last detection in humans is a noteworthy result, raising the question of potential unidentified viral reservoirs.

COASY related pontocerebellar hypoplasia type 12: A common Indian mutation with expansion of the phenotypic spectrum.

Pontocerebellar hypoplasia (PCH) type 12 is a rare, perinatal lethal neurodegenerative genetic disorder caused by biallelic mutations in the COASY gene. Herein, we describe the clinical and neuroradiological profile of nine affected fetuses/neonates from five families identified with a common COASY: c.1486-3C>G biallelic variant. Four of the five families were identified after data reanalysis of unresolved, severe PCH like phenotype and the fifth family through collaboration. The common antenatal phenotype was cerebellar hypoplasia. Microcephaly, arthrogryposis, and intrauterine growth restriction were the shared postnatal findings. The neurological manifestations included seizures, poor sucking, and spasticity. Novel findings of corpus callosum agenesis, simplified gyral pattern, normal sized pons, optic neuropathy, and a small thorax are reported in this series. The allele frequency of the COASY: c.1486-3C>G variant was 0.62% in the available Asian Indian database. We describe this as a possible common Indian origin variant. To the best of our knowledge, this is the largest PCH12 series reported.

Suleiman-El-Hattab syndrome: a histone modification disorder caused by TASP1 deficiency.

BACKGROUND: TASP1 encodes an endopeptidase activating histone methyltransferases of the KMT2 family. Homozygous loss-of-function variants in TASP1 have recently been associated with Suleiman-El-Hattab syndrome. We report six individuals with Suleiman-El-Hattab syndrome and provide functional characterization of this novel histone modification disorder in a multi-omics approach. METHODS: Chromosomal microarray/exome sequencing in all individuals. Western blotting from fibroblasts in two individuals. RNA sequencing and proteomics from fibroblasts in one individual. Methylome analysis from blood in two individuals. Knock-out of tasp1 orthologue in zebrafish and phenotyping. RESULTS: All individuals had biallelic TASP1 loss-of-function variants and a phenotype including developmental delay, multiple congenital anomalies (including cardiovascular and posterior fossa malformations), a distinct facial appearance and happy demeanor. Western blot revealed absence of TASP1. RNA sequencing/proteomics showed HOX gene downregulation (HOXA4, HOXA7, HOXA1 and HOXB2) and dysregulation of transcription factor TFIIA. A distinct methylation profile intermediate between control and Kabuki syndrome (KMT2D) profiles could be produced. Zebrafish tasp1 knock-out revealed smaller head size and abnormal cranial cartilage formation in tasp1 crispants. CONCLUSION: This work further delineates Suleiman-El-Hattab syndrome, a recognizable neurodevelopmental syndrome. Possible downstream mechanisms of TASP1 deficiency include perturbed HOX gene expression and dysregulated TFIIA complex. Methylation pattern suggests that Suleiman-El-Hattab syndrome can be categorized into the group of histone modification disorders including Wiedemann-Steiner and Kabuki syndrome.

Temporal Increase in Neutralization Potency of SARS-CoV-2 Antibodies and Reduced Viral Variant Escape after Sputnik V Vaccination

Recent studies have shown a temporal increase in the neutralizing antibody potency and breadth to SARS-CoV-2 variants in coronavirus disease 2019 (COVID-19) convalescent individuals. Here, we examined longitudinal antibody responses and viral neutralizing capacity to the B.1 lineage virus (Wuhan related), to variants of concern (VOCs: Alpha, Beta, Gamma, and Delta) and a local variant of interest (VOI: Lambda) in volunteers receiving the Sputnik V vaccine in Argentina. A collection of 472 serum samples obtained between January and September 2021 was used. The analysis indicates that while anti-spike IgG levels significantly wane over time, the neutralizing capacity to the first-wave linages of SARS-CoV-2 and VOC are maintained within four months of vaccination. In addition, an improved antibody cross-neutralizing ability to circulating variants of concern (Beta, Gamma and Delta) was observed over time of vaccination. The viral variants that displayed higher escape to neutralizing antibodies with respect to the original virus (Beta and Gamma variants) were the ones showing the largest increase in susceptibility to neutralization over time after vaccination. Our observations indicate that serum neutralizing antibodies are maintained for at least four month and show a reduction of VOC escape over time of vaccination.

Successful application of genome sequencing in a diagnostic setting: 1007 index cases from a clinically heterogeneous cohort.

Despite clear technical superiority of genome sequencing (GS) over other diagnostic methods such as exome sequencing (ES), few studies are available regarding the advantages of its clinical application. We analyzed 1007 consecutive index cases for whom GS was performed in a diagnostic setting over a 2-year period. We reported pathogenic and likely pathogenic (P/LP) variants that explain the patients' phenotype in 212 of the 1007 cases (21.1%). In 245 additional cases (24.3%), a variant of unknown significance (VUS) related to the phenotype was reported. We especially investigated patients which had had ES with no genetic diagnosis (n = 358). For this group, GS diagnostic yield was 14.5% (52 patients with P/LP out of 358). GS should be especially indicated for ES-negative cases since up to 29.6% of them  could benefit from GS testing (14.5% with P/LP, n = 52 and 15.1% with VUS, n = 54). Genetic diagnoses in most of the ES-negative/GS-positive cases were determined by technical superiority of GS, i.e., access to noncoding regions and more uniform coverage. Importantly, we reported 79 noncoding variants, of which, 41 variants were classified as P/LP. Interpretation of noncoding variants remains challenging, and in many cases, complementary methods based on direct enzyme assessment, biomarker testing and RNA analysis are needed for variant classification and diagnosis. We present the largest cohort of patients with GS performed in a clinical setting to date. The results of this study should direct the decision for GS as standard second-line, or even first-line stand-alone test.

GLYT1 encephalopathy: Further delineation of disease phenotype and discussion of pathophysiological mechanisms.

GLYT1 encephalopathy is a form of glycine encephalopathy caused by disturbance of glycine transport. The phenotypic spectrum of the disease has not yet been completely described, as only four unrelated families with the disorder have been reported to date. Common features of affected patients include neonatal hypotonia, respiratory failure, encephalopathy, myoclonic jerks, dysmorphic features, and musculoeskeletal anomalies. All reported affected patients harbor biallelic genetic variants in SLC6A9. SNP array together with Sanger sequencing were performed in a newborn with arthrogryposis and severe neurological impairment. The novel genetic variant c.997delC in SLC6A9 was detected in homozygous state in the patient. At protein level, the predicted change is p.(Arg333Alafs*3), which most probably results in a loss of protein function. The variant cosegregated with the disease in the family. A subsequent pregnancy with ultrasound anomalies was also affected. The proband presented the core phenotypic features of GLYT1 encephalopathy, but also a burst suppression pattern on the electroencephalogram, a clinical feature not previously associated with the disorder. Our results suggest that the appearance of this pattern correlates with higher cerebrospinal fluid glycine levels and cerebrospinal fluid/plasma glycine ratios. A detailed discussion on the possible pathophysiological mechanisms of the disorder is also provided.

Emergency Response for Evaluating SARS-CoV-2 Immune Status, Seroprevalence and Convalescent Plasma in Argentina

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used to assess antibody titers for clinical trials and therapies across the country. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs. AUTHOR SUMMARYThe development of robust and specific serologic assays to detect antibodies to SARS-CoV-2 is essential to understand the pandemic evolution and to stablish mitigation strategies. Here, we report the emergency development, production and application of a versatile ELISA test for detecting antibodies against the whole spike protein and its receptor binding domain. Over half million tests have been freely distributed in public and private health institutions of Argentina for evaluating immune responses, convalescent plasma programs and for large seroprevalence studies in neighborhoods and health care workers. We are still learning how and when to use serologic testing in different epidemiological settings. This program allowed us to produce large amount of high quality data on antibody levels in symptomatic and asymptomatic SARS-CoV-2 infections and generate relevant information about IgM and IgG seroconversion time and kinetics. We also present standardized protocols for antibody quantification as guidance for convalescent donor plasma selection in hospitals throughout the country for compassionate use and clinical trials. Here, we provide a framework for generating widely available tools, protocols and information of antibody responses for pandemic management.

Decreased Esophageal Sensitivity to Acid in Morbidly Obese Patients: A Cause for Concern?

BACKGROUND/AIMS: To evaluate esophageal sensitivity to acid between morbidly obese (MO) patients and non-MO controls with abnormal esophageal acid exposure. METHODS: We conducted a cross-sectional study of 58 patients: 30 MO (cases) and 28 non-MO (controls). Esophageal symptoms and esophageal sensitivity to 0.1 M hydrochloric acid solution (Bernstein test) were compared between MO and non-MO patients with a prior diagnosis of abnormal esophageal acid exposure. RESULTS: MO patients were less symptomatic than non-MO controls (14% vs 96%; odds ratio [OR], 0.006; 95% confidence interval [CI], 0.001 to 0.075; p=0.000). MO patients were more likely to present with decreased esophageal sensitivity to the instillation of acid than non-MO controls (57% vs 14%; OR, 8; 95% CI, 1.79 to 35.74; p=0.009). Subgroup analysis revealed no differences in esophageal sensitivity in MO patients with and without abnormal esophageal acid exposure (43% vs 31%; p=0.707). CONCLUSIONS: Silent gastroesophageal reflux disease (GERD) is common among MO individuals, likely due to decreased esophageal sensitivity to acid. The absence of typical GERD symptoms in these patients may delay discovery of precancerous conditions, such as Barrett’s esophagus. We believe that these patients may require a more aggressive diagnostic work-up to rule out the presence of silent GERD.

Diagnóstico molecular de enfermedades genéticas: del diagnóstico genético al diagnóstico genómico con la secuenciación masiva / Molecular diagnosis of genetic diseases: from genetic to genomic diagnosis using next generation sequencing

En la actualidad se conocen 8.000 enfermedades genéticas monogénicas. La mayoría de ellas son heterogéneas, por lo que el diagnóstico molecular por técnicas convencionales de secuenciación suele ser largo y costoso debido al gran número de genes implicados. El tiempo estimado para el diagnóstico molecular se encuentra entre 1 y 10 años, y este retraso impide que los pacientes reciban medidas terapéuticas y de rehabilitación específicas, que sus familiares entren en programas preventivos y que reciban asesoramiento genético. La secuenciación masiva está cambiando el modelo de diagnóstico molecular de los afectos, sin embargo, los médicos y profesionales de la salud se enfrentan al dilema de la selección del método más eficiente, con el menor coste sanitario y con la mayor precisión de sus resultados. El objetivo de este trabajo es revisar la tecnología de secuenciación masiva y definir las ventajas y los problemas en su utilización.

Currently 8000 monogenic genetic diseases are known. Most of them are heterogeneous, so their molecular diagnosis by conventional sequencing techniques is labour intensive and time consuming due to the large number of genes involved. The estimated time is between 1 and 10 years for molecular diagnosis and this delay prevents patients from receiving therapy and rehabilitation measures, and their families from entering prevention programs and being given genetic counselling. Next generation sequencing (NGS) is changing the model of molecular diagnosis of patients; however, doctors and health professionals are faced with the dilemma of choosing the most efficient method, with lower health care costs and the most accurate results. The aim of this paper is to review the NGS technology and define the advantages and problems in the use of this technology.

Evaluating DNA methylation and gene expression variability in the human term placenta.

UNLABELLED: Obtaining representative samples from a term placenta for gene-expression studies is confounded by both within placental heterogeneity and sampling effects such as sample location and processing time. Epigenetic processes involved in the regulation of gene expression, such as DNA methylation, may show similar variability, but are less well studied. Therefore, we investigated the nature of within and between- placenta variation in gene expression and DNA methylation of genes that were chosen for being differentially expressed or methylated by cell type within the placenta. METHODS: In total, two or more samples from each of 38 normal term placentae were utilized. The expression levels of CDH1, CDH11, ID2, PLAC1 and KISS1 were evaluated by real-time PCR. DNA methylation levels of LINE1 elements and CpGs within the promoter regions of KISS1, PTPN6, CASP8, and APC were similarly quantified by pyrosequencing. RESULTS: Despite considerable sample-to-sample variability within each placenta, the within-placenta correlation for both gene expression and methylation was significant for each studied gene. Most of this variability was not due to sample location. However, between placental differences in gene expression were inflated by the dramatic effect of processing time (0-24 h) on mRNA levels, particularly for PLAC1 and KISS1 (both expressed in the apical syncytiotrophoblast). In contrast, DNA methylation levels remained relatively constant over this same time period. CONCLUSION: Due to extensive site-to-site variability, multiple sampled sites are needed to accurately represent a placenta for molecular studies. Furthermore, mRNA quantitation of some genes may be hampered by its rapid degradation post-delivery (and possibly perinatally) and thus processing time should be considered in such analyses. Within-placenta correlations in expression and methylation from unrelated genes raise the possibility that methylation and expression variation may potentially reflect cell composition differences between samples rather than true differences occurring at the cellular level.

Identification of copy number variants in miscarriages from couples with idiopathic recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy loss (RPL), defined as two or more miscarriages, affects 3-5% of couples trying to establish a family. Despite extensive evaluation, no factor is identified in ∼40% of cases. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in miscarriages with normal karyotypes (46,XY or 46,XX) from couples with idiopathic RPL. METHODS: Array comparative genomic hybridization (array-CGH) was used to assess for DNA copy number variants (CNVs) in 26 miscarriages with normal karyotypes. Parental array-CGH analysis was performed to determine if miscarriage CNVs were de novo or inherited. RESULTS: There were 11 unique (previously not described) CNVs, all inherited, identified in 13 miscarriages from 8 couples. The maternal origin of two CNVs was of interest as they involved the imprinted genes TIMP2 and CTNNA3, which are only normally expressed from the maternal copy in the placenta. Two additional cohorts, consisting of 282 women with recurrent miscarriage (RM) and 61 fertile women, were screened for these two CNVs using a Quantitative Multiplex Fluorescent PCR of Short Fragments assay. One woman with RM, but none of the fertile women, carried the CTNNA3-associated CNV. CONCLUSIONS: This preliminary study shows that array-CGH is useful for detecting CNVs in cases of RPL. Further investigations of CNVs, particularly those involving genes that are imprinted in placenta, in women with RPL could be worthwhile.

Estudio colaborativo de abortos espontáneos: empleo de técnicas moleculares en muestras cuyo resultado citogenético no puede ser establecido / Colaborative study of spontaneus abortion: use of molecular techniques in failure culture samples

Introducción. El conocimiento de la causa de abortopermite ofrecer un adecuado consejo genético, además dereducir el estrés psicológico y el sentimiento de culpabilidadgenerado en la mujer tras sufrir un aborto. En la actualidadel estudio de abortos se realiza mediante técnicas citogenéticas,aunque muchas veces no es posible ofrecer un diagnósticodebido al fracaso de cultivo que acontece entre el5-42 % de los casos. En este trabajo se pretende evaluar lasensibilidad de las técnicas moleculares para el estudio dealteraciones cromosómicas en aquellos casos cuyo resultadocitogenético no puede ser establecido.Metodología. Se han estudiado 70 muestras de abortosdel primer y segundo trimestre de gestación mediante citogenéticay genética molecular (Quantitative FluorescentPolymerase chain Reaction [QF-PCR] y Multiplex ligationdependentProbe Amplification [MLPA]).Resultados. No se pudo establecer un resultado citogenéticoen el 37,2 % de las muestras. 44 fueron cariotipadas,obteniéndose 37 cariotipos normales y 7 anómalos. El estudiomolecular permitió establecer una dotación cromosómicaen el 100% de los casos, se encontraron alteraciones en10, de los cuales 3 carecían de resultado citogenético.Conclusiones. Se propone incluir en el protocolo de estudiode abortos el empleo de las técnicas citadas en casode fracaso del cultivo celular. La colaboración entre laboratoriosde citogenética y genética molecular especializados,permitiría ofrecer un resultado a la mayoría de las pacientes,esencial a la hora de poder establecer un adecuado consejogenético(AU)

Introduction. The knowledge of miscarriage causepermits offering an appropriate genetic counselling andmoreover, reduces psychological distress and self blamefeelings in women with a miscarriage. Nowadays, chromosomalstudy of abortions is mostly performed usingcytogenetic techniques. In few cases, no diagnosis can beestablished due to the high rate of culture failure (5-42%). Here, we try to evaluate the usefulness of moleculartechniques for the chromosomal study of those casesin which a cytogenetic result can not be established.Methods. A total of 70 miscarriage samples from thefirst and second trimesters of gestation have been studiedby karyotyping and molecular techniques (QF-PCRy MLPA).Results. The karyotype could not be established in37.2% of cases. Karyotype could be obtained in 44 samples,being 37 normal and 7 chromosomally abnormal.Molecular studies permitted to obtain a result in 100% ofthe cases, finding abnormalities in 10 of them, including3 in which the karyotype had not been established.Conclusions. We propose the use of molecular techniquesin those cases in which the culture fails. The collaborationbetween cytogenetic and molecular laboratorieswould permit to establish a result in the majority ofcases, which would represent an improvement for the offeringof an appropriate genetic counselling(AU)

Estimación del consumo de recursos sanitarios de los trastornos depresivos en la Comunidad Autónoma de Madrid / Estimation of the consumption of health care resources of depressive disorders in the Comunidad Autónoma de Madrid

El presente estudio trata de realizar una estimación del consumo de recursos sanitarios y costes monetarios medios generados por pacientes de la Comunidad de Madrid (CAM) atendidos en Centros de Salud Mental (CSM) e identificar factores que pueden estar asociados con el coste total, además de estudiar la comorbilidad física y psiquiátrica. Para ello se realizó un estudio descriptivo transversal, siendo la población de referencia 309 pacientes de ambos sexos, mayores de 17 años, con diagnóstico CIE-9 de depresión neurótica, reacción depresiva breve, reacción depresiva prolongada y trastorno depresivo no clasificado en otra parte. El coste medio total por paciente es de 442,15e, el coste medio del consumo de fármacos es 378,58e y el gasto medio por paciente de las asistencias es de 63,58e. Existe una elevada variabilidad entre los costes mínimos y máximos que ha llevado a dividir la muestra en grupos mediante cuartiles de gastos para su análisis y se concluye que el diagnóstico con mayor porcentaje de pacientes es el de depresión neurótica, siendo el que presenta los mayores costes (546,14e) (AU)

The present study tries to make an estimation of the consumption of health care resources and monetary costs means generated by patients of the Community of Madrid (CAM) seen in Mental Health Centers (CSM) and to identify factors that may be associated with the total cost, beside studying the phisical and psychiatric conmorbidity. We conducted a corss-sectional study, being the reference population of 309 male and female patients older than 17 years, with ICD-9 diagnosis of depression neurotic, depressive reaction shortly prolonged depressive reaction and depressive disorder not classified under another part. The average totalcost per patient is 442,15 e the average cost of the drugs is 378,58 e and the average expense per patient attendance is 63,58 e. There is a high variability between the minimum and maximum costs that led to divide the sample into groups by quartile expenses for analysis and concluded that the diagnosis of patients with the highest percentage is the neurotic depression, and the one that presents the greatest costs (546,14 e) (AU)

Protocolo recomendado para el estudio cromosómico de abortos espontáneos: abordaje citogenético y molecular / Recommended protocol for the chromosomal study of spontaneous miscarriages: cytogenetic and molecular approach

Las alteraciones cromosómicas son responsables de más del 60 % de las pérdidas embrionarias que acontecen durante el primer trimestre de gestación. El diagnóstico de la causa del aborto reduce significativamente tanto el estrés psicológico a largo plazo como el sentimiento de culpabilidad generado en la mujer tras sufrir un aborto y permite ofrecer un adecuado consejo genético a estas parejas de cara a futuros embarazos. Tradicionalmente, el estudio cromosómico de los abortos espontáneos se ha abordado mediante el cultivo celular de los restos fetales para su posterior cariotipado. Sin embargo, el estudio citogenético de los restos abortivos en particular presenta determinadas limitaciones, como la elevada tasa de fracaso del cultivo celular o el sobrecrecimiento de células de origen materno en el cultivo. El empleo de técnicas moleculares como la QF-PCR y MLPA permite obtener información acerca de la constitución cromosómica del aborto solventando tales limitaciones. En el presente trabajo se propone el protocolo óptimo para el estudio cromosómico de los restos abortivos incluyendo un abordaje citogenético y molecular

Chromosomal abnormalities account for at least 60 % offirst trimester fetal losses. Identification of the cause of fetal loss significantly reduces long-term psychological distress and self-blame feelings in women with a miscarriage and enables offering an improved genetic counselling to these couples in future pregnancies. Routine cytogenetic study of miscarriagesentails high rates of culture failure or misdiagnosis due to maternal cell contamination. On the other hand, molecular techniques such as QF-PCR and MLPA have been proved to be reliable methods to obtain information about chromosomal constitution of miscarriages, solving culture related limitations. In the present manuscript, we propose the optimal protocol for the chromosomal study of spontaneous abortions from a combined cytogenetic and molecular approach

Improvement in strategies for the non-invasive prenatal diagnosis of Huntington disease.

PURPOSE: We focused on the improvements of prenatal diagnosis by the analysis of DNA from maternal plasma, using Huntington disease as a model of disease. METHODS: We studied plasma from a pregnancy at risk of having a fetus affected with Huntington disease by the use of two direct analysis of the mutation and polymorphic STRs. RESULTS: Direct methods were not informative. Analysis with STRs revealed the presence of the allele that does not co-segregate with the disease, thus the fetus was healthy. CONCLUSIONS: This strategy is very useful to face complex cases when the direct study is not informative not only for Huntington disease but also for many other disorders.

A small and active ring X chromosome in a female with features of Kabuki syndrome.

A ring X chromosome is found in about 6% of patients with Turner syndrome (TS), often with mosaicism for a 45,X cell line. Patients with this karyotype are reported to have a higher incidence of a more severe phenotype including mental retardation. In fact, some studies have shown a correlation between this severity and the presence or absence of an intact and functional X inactivation center (XIST). However, the phenotype of the individuals with r(X) cannot be entirely defined in terms of their X-inactivation patterns. Nevertheless, a small group of these patients have been described to manifest clinical features reminiscent of the Kabuki syndrome. Here we present a female patient with clinical features resembling Kabuki syndrome and a mos 45,X/46,X,r(X) karyotype. Methylation analyses of polymorphic alleles of the androgen receptor gene showed that both alleles were unmethylated suggesting an active ring chromosome. A specific X chromosome array CGH was performed estimating the size of the ring to be 17 Mb, lacking the XIST gene, and including some genes with possible implications in the phenotype of the patient.

Early noninvasive prenatal detection of a fetal CRB1 mutation causing Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is one of the most severe inherited retinal dystrophies with the earliest age of onset. Mutations in the Crumbs homologue 1 (CRB1; OMIM 600105) gene explain 10%-24% of cases with LCA depending on the population. The aim of the present work was to study a fetal mutation associated to LCA in maternal plasma by a new methodology in the noninvasive prenatal diagnosis field: the denaturing High Performance Liquid Chromatography (dHPLC). METHODS: This study presents the case of a compound heterozygous fetus for two mutations in CRB1 (1q3.1-q32.2). dHPLC and automated DNA sequencing were used to detect the paternally inherited fetal mutation in a maternal plasma sample collected at the 12th week of gestation. To test the detection limit of dHPLC, we made serial dilutions of paternal DNA in control DNA. RESULTS: We were able to detect the presence of the paternally inherited fetal CRB1 mutation in maternal plasma by dHPLC. Moreover, by comparing chromatograms of serial dilutions to the plasma sample, we could ascertain that the percentage of fetal DNA in maternal plasma was at least 2%. However, the detection of the fetal mutation was not possible by automated DNA sequencing. CONCLUSIONS: dHPLC seems to be sensitive enough to detect small amounts of fetal DNA in maternal plasma samples. It could be a useful tool for the noninvasive prenatal detection of paternally inherited point mutations associated with retinopathies.

New strategy for the prenatal detection/exclusion of paternal cystic fibrosis mutations in maternal plasma.

BACKGROUND: Since the presence of fetal DNA was discovered in maternal blood, different investigations have focused on non-invasive prenatal diagnosis. The analysis of fetal DNA in maternal plasma may allow the diagnosis of fetuses at risk of cystic fibrosis (CF) without any risk of fetal loss. Here, we present a new strategy for the detection of fetal mutations causing CF in maternal plasma. METHODS: We have used a mini-sequencing based method, the SNaPshot, for fetal genotyping of the paternal mutation in maternal blood from three pregnancies at risk of CF. RESULTS: The paternal mutation was detected in the analysis of plasma samples from cases 1 and 3 but not in case 2. Results of a posterior conventional molecular analysis of chorionic biopsies were in full agreement with those obtained from analysis of the plasma samples. CONCLUSIONS: The knowledge about the inheritance of the paternal mutation in a fetus may avoid the conventional prenatal diagnosis in some cases. The SNaPshot technique has been shown to be a sensitive and accurate method for the detection of fetal mutations in maternal plasma. Its ease handling, rapid and low cost makes it appropriate for a future routine clinical use in non-invasive prenatal diagnosis of cystic fibrosis.

New type of mutations in three spanish families with choroideremia.

PURPOSE: Choroideremia (CHM) is an X-linked ophthalmic disease. The gene associated with CHM (REP-1) encodes a ubiquitously expressed protein that is indispensable for the posttranslational activation of retina-specific Rab protein. Different mutations, including large genomic rearrangements involving the REP-1 gene, are responsible for CHM, but they all cause the protein to be truncated or absent. The authors screened 20 Spanish families with clinical diagnoses of CHM to determine the molecular cause of the disease. METHODS: First, the authors performed haplotype analyses to determine whether the disease is linked to the REP-1 gene. In families in whom the disease segregated with the CHM locus (n = 14), mutational screening of the REP-1 gene was performed. RESULTS: In 13 of the 14 families in which the phenotype segregated with the CHM locus, the authors identified the mutation associated with the disease. Eight different molecular defects that led to truncation and one that led to complete absence of the REP-1 protein were found in nine families and one family, respectively. Furthermore, the authors identified a novel type of mutation in the REP-1 gene in three families. This novel type of mutation did not result in a truncated or absent protein. Rather, these patients lost different parts of the REP-1 mRNA in-frame that in all the cases encode a conserved protein domain implicated in the interaction with Rab proteins. CONCLUSIONS: Based on the different mutations found, the authors propose a four-step protocol for the molecular diagnosis of CHM.