Lessons from two series by physicians and caregivers' self-reported data in DDX3X-related disorders.

INTRODUCTION AND METHODS: We report two series of individuals with DDX3X variations, one (48 individuals) from physicians and one (44 individuals) from caregivers. RESULTS: These two series include several symptoms in common, with fairly similar distribution, which suggests that caregivers' data are close to physicians' data. For example, both series identified early childhood symptoms that were not previously described: feeding difficulties, mean walking age, and age at first words. DISCUSSION: Each of the two datasets provides complementary knowledge. We confirmed that symptoms are similar to those in the literature and provides more details on feeding difficulties. Caregivers considered that the symptom attention-deficit/hyperactivity disorder were most worrisome. Both series also reported sleep disturbance. Recently, anxiety has been reported in individuals with DDX3X variants. We strongly suggest that attention-deficit/hyperactivity disorder, anxiety, and sleep disorders need to be treated.

Missense mutations of conserved glycine residues in fibrillin-1 highlight a potential subtype of cb-EGF-like domains.

In six index cases/families referred for Marfan syndrome (MFS) molecular diagnosis, we identified six novel mutations in the FBN1 gene: c.1753G>C (p.Gly585Arg), c.2456G>A (p.Gly819Glu), c.4981G>A (p.Gly1661Arg), c.5339G>A (p.Gly1780Glu), c.6418G>A (p.Gly2140Arg) and c.6419G>A (p.Gly2140Glu). These variants, predicted to result in Glycine substitutions are located at the third position of a 4 amino acids loop-region of calcium-binding Epidermal Growth Factor-like (cb-EGF) fibrillin-1 domains 5, 9, 24, 25 and 32. Familial segregation studies showing cosegregation with MFS manifestations or de novo inheritance in addition to in silico analyses (conservation, 3D modeling) suggest evidence for a crucial role of the respective Glycine positions. Extending these analyses to all Glycine residue at position 3 of this 4 residues loop in fibrillin-1 cb-EGF with the UMD predictor tool and alignment of 2038 available related sequences strongly support a steric strain that only allows Glycine or even Alanine residues for domain structure maintenance and for the fibrillin functions. Our data compared with those of the literature strongly suggest the existence of a cb-EGF domain subtype with implications for related diseases.

First molecular screening of deafness in the Altai Republic population.

BACKGROUND: We studied the molecular basis of NSHL in Republic of Altai (South Siberia, Russia). The Altaians are the indigenous Asian population of the Altai Mountain region considered as a melting-pot and a dispersion center for world-wide human expansions in the past. METHODS: A total of 76 patients of Altaian, Russian or mixed ethnicity and 130 Altaian controls were analyzed by PCR-DHPLC and sequencing in the GJB2 gene. The GJB6 deletion and the common non-syndromic deafness-causing mitochondrial mutations were also tested when appropriate. RESULTS: 8.3% of the Altaian chromosomes were carrying GJB2 mutations versus 46.9% of the Russian chromosomes. The 235delC mutation was predominant among Altaians, whereas the 35delG mutation was most prevalent among Russian patients. CONCLUSION: We found an Asian-specific GJB2 diversity among Altaians, and different GJB2 contribution for deafness in the Altaian and Russian patients. The high carrier frequency of 235delC in Altaians (4.6%) is probably defined by gene drift/founder effect in a particular group. The question whether the Altai region could be one of founder sources for the 235delC mutation widespread in Asia is open.

A large deletion including most of GJB6 in recessive non syndromic deafness: a digenic effect?

Congenital profound deafness has a known genetic origin in more than 50% of all cases. The majority of the non syndromic hearing loss (NSHL) show an autosomal recessive inheritance. Mutations in the GJB2 gene (connexin 26) account for more than 50% of the recessive non syndromic deafness (DFNB1) among 30 loci. Other connexin genes have been more rarely involved and attention was given here to the GJB6 gene (connexin 30). We show that homozygous deletion of a minimal 150 kb region encompassing this gene causes NSHL. More strikingly, association of this deletion in trans of the GJB2 gene 35delG or E47X mutations is also associated with NSHL.

Molecular epidemiology of DFNB1 deafness in France.

BACKGROUND: Mutations in the GJB2 gene have been established as a major cause of inherited non syndromic deafness in different populations. A high number of sequence variations have been described in the GJB2 gene and the associated pathogenic effects are not always clearly established. The prevalence of a number of mutations is known to be population specific, and therefore population specific testing should be a prerequisite step when molecular diagnosis is offered. Moreover, population studies are needed to determine the contribution of GJB2 variants to deafness. We present our findings from the molecular diagnostic screening of the GJB2 and GJB6 genes over a three year period, together with a population-based study of GJB2 variants. METHODS AND RESULTS: Molecular studies were performed using denaturing High Performance Liquid Chromatograghy (DHPLC) and sequencing of the GJB2 gene. Over the last 3 years we have studied 159 families presenting sensorineural hearing loss, including 84 with non syndromic, stable, bilateral deafness. Thirty families were genotyped with causative mutations. In parallel, we have performed a molecular epidemiology study on more than 3000 dried blood spots and established the frequency of the GJB2 variants in our population. Finally, we have compared the prevalence of the variants in the hearing impaired population with the general population. CONCLUSION: Although a high heterogeneity of sequence variation was observed in patients and controls, the 35delG mutation remains the most common pathogenic mutation in our population. Genetic counseling is dependent on the knowledge of the pathogenicity of the mutations and remains difficult in a number of cases. By comparing the sequence variations observed in hearing impaired patients with those sequence variants observed in general population, from the same ethnic background, we show that the M34T, V37I and R127H variants can not be responsible for profound or severe deafness.

Nonsyndromic 35 delG mutation of the connexin 26 gene associated with deafness in syndromic children: two case reports.

OBJECTIVES/HYPOTHESIS: Several genetic diseases, such as velocardiofacial syndrome Del(22q11) and Down syndrome, are associated with hearing impairment. STUDY DESIGN: Case reports. METHODS: The authors reported two cases of hearing-impaired children, one with Del (22q11) and one with Down syndrome, both with bilateral nonevolutive profound sensorineural deafness. Because of unusual features of their deafness and familial history, genetic evaluation was proposed. A homozygous 35delG mutation on the Connexin 26 gene was found in both children (DFNB1 phenotype). RESULTS: A review of the reported otological features of Del (22q11) and Down syndrome showed that sensorineural deafness is rare and seldom profound. The authors found no evidence for a genetic link between Del(22q11) or Down syndrome and 35delG mutation on the Connexin 26 gene. CONCLUSION: The case reports reveal a coincidental association between DFNB1 and a multiple congenital anomaly syndrome. The clinician must be aware of this type of association to manage genetic counseling, appropriate otological care, and suitable treatment.

Comparación entre las técnicas de citocentrifugado y base líquida SurePathTM para procesamiento de muestras no ginecológicas / Comparison between cytocentrifugation techniques and liquid-based SurePathTM for the processing of non-gynaecological specimens

Objetivo: Evaluar el uso de base líquida de manera rutinaria para muestras no ginecológicas en nuestro laboratorio mediante una verificación de la validación y comparación de características morfológicas y la concordancia diagnóstica entre dos técnicas: citología convencional por citocentrífugado (CS) y citología de base líquida SurePathTM (CBL). Metodología: Consecutivamente se procesaron un total de 109 muestras no ginecológicas por las dos técnicas, usando una sola muestra dividida, la mitad de la muestra para cada técnica. Todas las láminas fueron revisadas por el mismo citopatólogo evaluando: celularidad, preservación, presencia de elementos que oscurecen (inflamación, hemorragia, moco, otros) y presencia de grupos de diagnóstico para cada categoría de diagnóstico final. Retrospectivamente se realizó una evaluación de conformidad con la biopsia de seguimiento cuando estaba disponible. Resultados: Se observó una concordancia buena por categoría diagnóstica en el 84% de las muestras, con un índice Kappa bueno (0,65). La proporción en categorías: negativo, atípicos y positivas fue del 69%, 18% y 11% para citología de base líquida y el 83%, 7% y 9% para citología convencional por citocentrífugado. Conclusión: La citología de base líquida es una técnica equiparable a la citología convencional por citocentrífugado (concordancia diagnóstica buena, índice kappa 0,6) y es superior en calidad ya que presenta una preservación inmediata de la muestra, un fondo limpio y menos elementos que oscurecen permitiendo un mejor examen morfológico.

Objective: The objective of this study was to evaluate the routine use of liquid based cytology for non-gynecologic specimens in our laboratory, comparing morphological characteristics and diagnostic agreement between the two techniques: conventional cytology cytospin, and SurePath liquid-based cytology. Study design: A total of 109 consecutive non-gynecologic specimens were processed, split into two, and then prepared using the two techniques. All slides were reviewed by the same cytopathologist, who evaluated: cellularity, preservation, obscuring elements (inflammation, bleeding, mucus, etc.), and presence of diagnostic groups for each final diagnostic category. When available, an evaluation of the biopsy was performed retrospectively. Results: Good agreement was observed by diagnostic category in 84% of specimens, with a good Kappa index (0.65). The proportion for each category: negative, atypical, and positive was 69%, 18%, and 11%, respectively, for cases processed by liquid-based cytology and 83%, 7%, and 9%, respectively, for conventional cytology cytospin. Conclusion: Liquid based cytology is equivalent to conventional cytology cytospin when cases are grouped by category (good agreement, kappa index 0.651), and is superior in quality because the specimen is well preserved, has a clean background and fewer obscuring elements allowing a better morphological examination.

Combined mutation and rearrangement screening by quantitative PCR high-resolution melting: is it relevant for hereditary recurrent Fever genes?

The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.