Técnicas para el diagnóstico molecular de enfermedades hereditarias / Techniques for molecular diagnosis of hereditary diseases

El diagnóstico de muchas enfermedades hereditarias necesita una confirmación a nivel molecular del defecto genético que presenta el paciente. Una vez detectada la mutación y confirmado el diagnóstico clínico, podemos determinar cuál es el efecto de dicha mutación en la proteína codificada(cambio de conformación, alteración o pérdida defunción, localización errónea, disminución en su expresión, etc.), y así poder abrir puertas a nuevas terapias dirigidas al defecto específico. En esta revisión, primero consideraremos algunos conceptos básicos de genética molecular, incluyendo estructura y expresión del gen, intrones y exones, códigos genéticos, transcripción, traducción, procesamiento del RNA y mutaciones y sus tipos. También explicaremos algunos métodos para extraer DNA y RNA de sangre u otros tejidos del paciente. A continuación discutiremos los métodos que se emplean para detectar mutaciones en genes asociados a enfermedades hereditarias. El principio en el que se basa un ensayo de detección de mutaciones es que la secuencia de nucleótidos del gen de un individuo afecto será distinta de la secuencia de un individuo con fenotipo normal. Además, describiremos dos métodos para analizar el efecto de algunas mutaciones en la maduración del pre-mRNA (RT-PCR a partir de RNA de sangre y análisis de minigenes). Por último, mencionaremos algunos métodos informáticos que sirven para determinar si las mutaciones detectadas son patológicas o no, y para predecir el efecto de mutaciones en la maduración del pre-mRNA (AU)

The diagnoses of many hereditary diseases must be confirmed on a molecular level of a genetic defect that the patient has. Once the mutation is detected and the clinical diagnoses confirmed, we can determine which is the effect of said mutation in the coded protein (changing shape, alteration or loss of function, erroneous location, decrease of its expression, etc.) and just be able to open the doors to new therapies aimed at the specific defect. In this article, we first consider some basic concepts of molecular genetics, including the gene structure and expression, introns and exons, genetic codes, transcription, translation, RNA processing, and mutations and its types. We will also give some explanations of methods to extract DNA and RNA from the blood and other tissues of the patient. After that, we will discuss the methods used to detect mutations in genes associated to hereditary diseases. The principal on which a mutation detection trial is based is that this sequence of gene nucleotides of an individual affected will be different from the sequence of an individual with normal phenotype. In addition, we will describe two methods to analyze the effect of some mutations in the maturation of pre-mRNA(RT-PCR from RNA of the blood and analysis of minigenes). Finally, we will mention some computer methods that serve to determine if the mutations detected are pathological or not and to predict the effect of the mutations in the maturation of the pre-mRNA (AU)

Molecular analysis of the CLCN5 gene in Dent's disease: first mutation identified in a patient from South America.

BACKGROUND: Dent's disease is a rare renal tubular disorder characterized by low-molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and eventual renal failure. The selective loss of low-molecular weight proteins points to a defect of the proximal tubule, where filtered proteins are normally reabsorbed by endocytosis. The disease tends to present in childhood or early adult life, and males are more severely affected than females. The disease is caused by mutations in CLCN5 or OCRL1, both on the X chromosome, which code for the chloride/proton exchange transporter ClC-5 and the phosphatidylinositol-4,5-biphosphate-5-phosphatase, respectively. METHODS: Mutational analysis of CLCN5 gene from 4 unrelated patients diagnosed with Dent's disease and their relatives, 3 from Spain and 1 from Bolivia, was performed by PCR and automatic DNA sequencing. RESULTS: In the current study, we report the identification of 4 mutations in CLCN5 of 1 family from Bolivia and 3 families from Spain. Two of the mutations are novel and consist of 1 nonsense mutation, Y502X, and 1 missense mutation, L225P, affecting ClC-5alpha-helix F, one of the helices involved in formation of the chloride selectivity filter. CONCLUSIONS: Our results add to the expanding spectrum of mutations in CLCN5. This is the first report of a CLCN5 mutation in a Dent's disease patient of South American origin.

Enfermedad de Bartter con sordera neurosensorial (Bartter tipo IV). Una entidad descrita hace solo diez años / Bartter disease with neurosensitive deafness (Bartter tpe IV). A clinical entity described 10 years ago

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Condrocalcinosis e hipomagnesemia en un paciente portador de una nueva mutación en el gen del cotransportador de ClNa sensible a tiazidas / Chondrocalcinosis and hypomagnesaemia in a patient with a new mutation in the gene of the thiazide-sensitive NA-Cl cotransporter

Se comunica la historia clínica de un paciente adulto que debutó en la adolescencia con episodios agudos de inflamación, monoarticulares, con afectación de grandes articulaciones de miembros superiores e inferiores, en ausencia de síntomas renales. El estudio radiológico mostró calcificaciones de los meniscos de ambas rodillas, de la sínfisis del pubis y de otras articulaciones. Al demostrarse la existencia de hipomagnesemia, se observó que tenía, además, hipopotasemia, hipocalciuria, incremento de la eliminación urinaria de magnesio, niveles de renina ligeramente elevados, defecto de dilución y una reducción moderada de la reabsorción de ClNa tubular distal renal, todo ello compatible con el diagnóstico de síndrome de Gitelman. La secuenciación de los exones y de las regiones intrónicas flanqueantes del gen SLC12A3, que codifica el cotransportador de ClNa sensible a tizaidas, mostró que el paciente es portador homocigótico de una nueva mutación en el intrón 7. La mutación consiste en el cambio de una G a una A en el sitio donador del "splicing" del RNA (AU)

Enfermedad de Dent. Historia y causas genéticas de una «nueva» tubulopatía / Dent disease. History and genetic causes of a «new» tubulopathy

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[Chondrocalcinosis and hypomagnesaemia in a patient with a new mutation in the gene of the thiazide-sensitive Na-Cl cotransporter]. / Condrocalcinosis e hipomagnesemia en un paciente portador de una nueva mutación en el gen del cotransportador de ClNa sensib e a tiazidas.

We report the clinical history of an adult patient that debuted during adolescence with sharp episodes of arthritis several joints, with affectation of big articulations of superior and inferior members, in absence of renal symptoms. An X-ray showed calcification of the menisci in both knees, the symphysis of the pubis and of other joints. When the presence of hypomagnesaemia was demonstrated, we observed that he also had hypokalemia, hypocalciuria, increment of the urinary elimination of magnesium, slightly high levels of renin, dilution defect and a moderate reduction of the NaCl tubular distal reabsorption, all compatible with the diagnosis of Gitelman syndrome. We sequenced the exons and intron flanking regions of the SLC12A3 gene, encoding the thiazide-sensitive Na-Cl cotransporter, and showed that the patient is homozygous for a new mutation in intron 7. This mutation consisted of a G to A transition at position +1 of the donor splice site of intron 7.

Mupirocin resistance in methicillin-resistant Staphylococcus aureus clinical isolates in a Spanish hospital. Co-application of multiplex PCR assay and conventional microbiology methods.

A total of 1785 Staphylococcus aureus clinical isolates were collected in our hospital during 1998 (526), 1999 (564) and 2000 (695). Among them, one hundred and thirty (39, 33 and 58, respectively) were phenotypically assigned as methicillin-resistant Staphylococcus aureus (MRSA); sixteen of these isolates (3, 2 and 11, respectively) were detected as highly mupirocin-methicillin resistant Staphylococcus aureus (MMRSA). In this work, our goal was to characterize MRSA and MMRSA clinical isolates by co-application of phenotypic and genotypic methods in order to determine the MMRSA incidence in our hospital during the period 1998-2000. With this purpose we compared and integrated the results obtained using conventional microbiology methods with those obtained using a multiplex polymerase chain reaction (PCR) assay. Our results showed a good complementation between these two approximations to determine the incidence of MMRSA clinical isolates and permitted to estimate that such incidence increased from 7.7% in 1998 to 19% in 2000.

A PCR assay for rapid detection of vancomycin-resistant enterococci.

Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.

Multiplex PCR for simultaneous identification of Staphylococcus aureus and detection of methicillin and mupirocin resistance.

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance), ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S. aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S. aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.

Enfermedad de Bartter neonatal diagnosticada mediante la detección de una mutación en el gen KCNJ1 que codifica la síntesis del canal renal de potasio ROMK1 / Neonatal bartter’s disease diagnosed by detecting a mutation in the kcnj1 gene which codes for the renal potassium channel ROMK1

Se comunican los datos clínicos de una paciente diagnosticada de síndrome de Bartter aunque, inicialmente se sospechó que estuviera afecta de hipercalciuria idiopática y, posteriormente, de síndrome de hiperprostaglandinismo E2. Fue producto de un embarazo pretérmino en el que se constató polihidramnios. Durante los dos primeros años de vida, a pesar de evidenciarse retraso ponderal importante, poliuria y tendencia especial a la deshidratación, no tenía hipopotasemia. El equilibrio ácido-base durante ese tiempo fue normal, salvo acidosis metabólica en los primeros días de vida. Tras apreciarse hipercalciuria, fue tratada contiazidas y dieta pobre en sal. Con este tratamiento se observó con frecuencia alcalosis hipopotasémica. Posteriormente, la posibilidad técnica de determinar los niveles de renina y de aldosterona y la eliminación urinaria de PGE2 permitió (..) (AU)

We report clinical data of a female patient with Bartter’s syndrome who was initially diagnosed with idiophatic hypercalciuria and, subsequently, with hyperprostaglandin E, syndrome. The patient was born after premature delivery with a history of polyhydramnios. During the first two years of life, in spite of evidence for significant failure to thrive, polyuria and special tendency to dehydration, she had no hypokalemia. The acid-base balance was normal except metabolic acidosis during the first few days after she was born. When hypercalciuria was observed, she was treated with thiazides and a low-salt diet. With such treatment she frequently showed hypokalemic alkalosis. Afterwards, once it was possible to determine the levels of renin and aldosterone and the urinary excretion of PGE2,we suspected the diagnosis. DNA sequencing analysis showed that the patient (..) (AU)

[Neonatal Bartter disease diagnosed with the detection of a mutation of the KCNJ1 gene which codifies the synthesis of the renal ROMK1 potassium channel]. / Enfermedad de Bartter neonatal diagnosticada mediante la detección de una mutación en el gen KCNJ1 que codifica la síntesis del canal renal de potasio ROMK1.

We report clinical data of a female patient with Bartter's syndrome who was initially diagnosed with idiophatic hypercalciuria and, subsequently, with hyperprostaglandin E, syndrome. The patient was born after premature delivery with a history of polyhydramnios. During the first two years of life, in spite of evidence for significant failure to thrive, polyuria and special tendency to dehydration, she had no hypokalemia. The acid-base balance was normal except metabolic acidosis during the first few days after she was born. When hypercalciuria was observed, she was treated with thiazides and a low-salt diet. With such treatment she frequently showed hypokalemic alkalosis. Afterwards, once it was possible to determine the levels of renin and aldosterone and the urinary excretion of PGE2, we suspected the diagnosis. DNA sequencing analysis showed that the patient carried a homozygotic mutation in the KCNJ1 gene, coding for the potassium channel ROMK, which results in the premature termination of the protein. It is the first time that this mutation has been found in Spain. The detection of this mutation confirmed a disease that was initially of uncertain diagnosis.

Glycopeptide resistance in enterococci.

The selective pressure resulting from the extensive use of antibiotics over the last 50 years has led to the emergence of bacterial resistance and to the dissemination of resistance genes among pathogenic microorganisms. Consequently, we are now at serious risk of suffering intractable, life-threatening infections. The progressive emergence and rapid dissemination of resistance to glycopeptides, the last resort for treating nosocomial infections with enterococci resistant to usual antibiotics, constitute one of the most dramatic examples of such resistance. Enterococci are normal human commensals, but are also a frequent cause of nosocomial urinary tract infections and nosocomial bacteremia. Enterococcus faecalis causes 80 to 90% of human enterococcal infections, while Enterococcus faecium accounts for most of the remainder. During the last decade, our understanding of the genetics and biochemical basis of resistance to glycopeptides has increased greatly. Furthermore, the application of molecular methods for the diagnosis of glycopeptide-resistant enterococci has provided new insights into the epidemiology of enterococcal infections.

Molecular cloning, tissue distribution, and chromosomal mapping of the human epithelial Ca2+ channel (ECAC1).

Functional and morphological analyses indicated that the epithelial Ca2+ channel (ECaC), which was recently cloned from rabbit kidney, exhibits the defining properties for being the gatekeeper in transcellular Ca2+ (re)absorption. Its human homologue provides, therefore, a molecular basis for achieving a better understanding of Ca2+ mal(re)absorption. By applying the RACE technique, the full-length cDNA of human ECaC (HGMW-approved symbol ECAC1) was obtained. It consisted of 2,772 bp with an open reading frame of 2,187 bp encoding a protein of 729 amino acids with a predicted molecular mass of 83 kDa. Phylogenetic analysis indicated that this highly selective Ca2+ channel exhibits a low level of homology (<30%) to other Ca2+ channels, suggesting that it belongs to a new family. hECaC was highly expressed in kidney, small intestine, and pancreas, and less intense expression was detected in testis, prostate, placenta, brain, colon, and rectum. These ECaC-positive tissues also expressed the 1,25-dihydroxyvitamin D3-sensitive calcium-binding proteins, calbindin-D9K and/or calbindin-D28K. The human ECaC gene mapped to chromosome 7q31.1-q31.2. Taken together, the conspicuous colocalization of hECaC and calbindins in organs that are not prime regulators of plasma Ca2+ levels could illustrate new pathways in cellular Ca2+ homeostasis.

Alternative splicing regulates the production of ARD-1 endoribonuclease and NIPP-1, an inhibitor of protein phosphatase-1, as isoforms encoded by the same gene.

ARD-1 is an endoribonuclease identified initially as the product of a human cDNA that complements mutations in rne, a gene that encodes Escherichia coli ribonuclease E. NIPP-1 was identified in bovine nuclear extracts as an inhibitor of protein phosphatase-1. Earlier work has shown that the protein-coding sequence of ARD-1 is identical to the carboxy-terminal third of NIPP-1. However, whether ARD-1 is present in eukaryotes as a distinct entity has been unclear, as neither ARD-1-specific transcripts nor ARD-1 protein were detected in mammalian cells in earlier studies. Here we show that ARD-1 exists in human cells as a discrete protein, and that the ARD-1 and NIPP-1 peptides are isoforms encoded by a single gene and the same alternatively spliced precursor RNA. A retained intron containing multiple translation stop codons that are configured to terminate translation and initiate nonsense-mediated decay, limits the production of cellular ARD-1 protein. Our results establish the process by which functionally disparate ARD-1 and NIPP-1 peptides are generated from the protein-coding sequence of the same gene in human cells.

Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors.

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that 'nuclear inhibitor of PP1' (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a 'dominant negative' mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.

DNA bending and the initiation of transcription at sigma54-dependent bacterial promoters.

We have examined the effects on transcription initiation of promoter and enhancer strength and of the curvature of the DNA separating these entities on wild-type and mutated enhancer-promoter regions at the Escherichia coli sigma54-dependent promoters glnAp2 and glnHp2 on supercoiled and linear DNA. Our results, together with previously reported observations by other investigators, show that the initiation of transcription on linear DNA requires a single intrinsic or induced bend in the DNA, as well as a promoter with high affinity for sigma54-RNA polymerase, but on supercoiled DNA requires either such a bend or a high affinity promoter but not both. The examination of the DNA sequence of all nif gene activator- or nitrogen regulator I-sigma54 promoters reveals that those lacking a binding site for the integration host factor have an intrinsic single bend in the DNA separating enhancer from promoter.

ARD-1 cDNA from human cells encodes a site-specific single-strand endoribonuclease that functionally resembles Escherichia coli RNase E.

The human ARD-1 (activator of RNA decay) cDNA sequence can rescue mutations in the Escherichia coli rne gene, which specifies the essential endoribonuclease RNase E, resulting in RNase E-like cleavages in vivo in rne-defective bacteria and in vitro in extracts isolated from these cells (Wang, M., and Cohen, S. N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10591-10595). Recent studies indicate that the 13.3-kDa protein encoded by ARD-1 cDNA is almost identical to the carboxyl-terminal end of the bovine protein NIPP-1, a nuclear inhibitor of protein phosphatase 1; separate transcripts formed by alternative splicing are proposed to encode the discrete ARD-1 and combined ARD-1/NIPP-1 products (Van Eynde, A., Wera, S., Beullens, M. , Torrekens, S., Van Leuven, F., Stalmans, W., and Bollens, M. (1995) J. Biol. Chem. 270, 28068-28074). Here we show that affinity column-purified protein encoded by human ARD-1 cDNA in E. coli is a site-specific Mg2+-dependent endoribonuclease that binds in vitro to RNase E substrates, cleaves RNA at the same sites as RNase E, and, like RNase E, generates 5' phosphate termini at sites of cleavage. Our results indicate that the ARD-1 peptide can function as a ribonucleolytic analog of E. coli RNase E as well as a domain of the protein phosphatase inhibitor, NIPP-1.