Autosomal recessive spinocerebellar ataxia SCAR8/ARCA1: first families detected in Spain.

INTRODUCTION: Autosomal recessive spinocerebellar ataxia type 8 (ARCA1/SCAR8) is caused by mutations of the SYNE1 gene. The disease was initially described in families from Quebec (Canada) with a phenotype of pure cerebellar syndrome, but in recent years has been reported with a more variable clinical phenotype in other countries. Cases have recently been described of muscular dystrophy, arthrogryposis, and cardiomyopathy due to SYNE1 mutations. OBJECTIVE: To describe clinical and molecular findings from 4 patients (3 men and one woman) diagnosed with ARCA1/SCAR8 from 3 Spanish families from different regions. MATERIAL AND METHODS: We describe the clinical, paraclinical, and genetic results from 4 patients diagnosed with ARCA1/SCAR8 at different Spanish neurology departments. RESULTS: Onset occurred in the third or fourth decade of life in all patients. After 15 years of progression, 3 patients presented pure cerebellar syndrome, similar to the Canadian patients; the fourth patient, with over 30 years' progression, presented vertical gaze palsy, pyramidal signs, and moderate cognitive impairment. In all patients, MRI studies showed cerebellar atrophy. The genetic study revealed distinct pathogenic SYNE1 mutations in each family. CONCLUSIONS: ARCA1/SCAR8 can be found worldwide and may be caused by many distinct mutations in the SYNE1 gene. The disease may manifest with a complex phenotype of varying severity.

Cerebellar Cognitive Affective Syndrome in Costa da Morte Ataxia (SCA36).

SCA36 is an autosomal dominant spinocerebellar ataxia (SCA) affecting many families from Costa da Morte, a northwestern region of Spain. It is caused by an intronic GGCCTG repeat expansion in NOP56. In order to characterize the cognitive and affective manifestations of this cerebellar disease, a group of 30 SCA36 mutation carriers (11 preataxic and 19 ataxic patients) were assessed with a comprehensive battery of standardized tests. Phonological verbal fluency - but not semantic fluency - was already mildly impaired in preataxic subjects. In ataxic patients, both phonological and semantic fluencies were significantly below normal. Depression, while more frequent and prominent in ataxic patients, was also often present in the preataxic stage. This is the first systematic study supporting the presence of a mild cerebellar cognitive and affective syndrome in SCA36. Routine evaluation of cognitive and emotional spheres in SCA36 patients as well as asymptomatic mutation carriers should allow early detection and timely therapeutic intervention.

Autosomal recessive spinocerebellar ataxia SCAR8/ARCA1: First families detected in Spain. / Heredoataxia cerebelosa recesiva ARCA1/SCAR8: primeras familias detectadas en España.

INTRODUCTION: Autosomal recessive spinocerebellar ataxia type 8 (ARCA1/SCAR8) is caused by mutations of the SYNE1 gene. The disease was initially described in families from Quebec (Canada) with a phenotype of pure cerebellar syndrome, but in recent years has been reported with a more variable clinical phenotype in other countries. Cases have recently been described of muscular dystrophy, arthrogryposis, and cardiomyopathy due to SYNE1 mutations. OBJECTIVE: To describe clinical and molecular findings from 4 patients (3 men and one woman) diagnosed with ARCA1/SCAR8 from 3 Spanish families from different regions. MATERIAL AND METHODS: We describe the clinical, paraclinical, and genetic results from 4 patients diagnosed with ARCA1/SCAR8 at different Spanish neurology departments. RESULTS: Onset occurred in the third or fourth decade of live in all patients. After 15 years of progression, 3 patients presented pure cerebellar syndrome, similar to the Canadian patients; the fourth patient, with over 30 years' progression, presented vertical gaze palsy, pyramidal signs, and moderate cognitive impairment. In all patients, MRI studies showed cerebellar atrophy. The genetic study revealed distinct pathogenic SYNE1 mutations in each family. CONCLUSIONS: ARCA1/SCAR8 can be found worldwide and may be caused by many distinct mutations in the SYNE1 gene. The disease may manifest with a complex phenotype of varying severity.

PLA2G6 mutations associated with a continuous clinical spectrum from neuroaxonal dystrophy to hereditary spastic paraplegia.

PLA2G6-associated neurodegeneration (PLAN) and hereditary spastic paraplegia (HSP) are 2 groups of heterogeneous neurodegenerative diseases. In this study, we report PLA2G6 gene mutations in 3 families from Turkey, Morocco, and Romania. Two affected Turkish siblings presenting HSP adds the disease to PLAN phenotypes. They were homozygous for the PLA2G6 missense c.2239C>T, p.Arg747Trp variant and the ages of onset were 9 and 21. Parkinsonism, dystonia or cognitive decline were not the clinical elements in these patients contrary to the cases that has been previously reported with the same variant, however, iron accumulation was evident in their cranial magnetic resonance imaging. The Moroccan patient was homozygous for a novel missense c.1786C>T, p.Leu596Phe variant and the Romanian patient had 2 novel mutations; c.1898C>T, p.Ala633Val and c.1765_1768del, p.Ser589ThrfsTer76. Both of these patients conformed better to childhood onset PLAN with the age of onset at 4 and 7 years, respectively. Interestingly, all identified mutations were affecting the highly conserved patatin-like phospholipase domain of the PLA2G6 protein.

Evaluating the Calling Performance of a Rare Disease NGS Panel for Single Nucleotide and Copy Number Variants.

INTRODUCTION: Variant detection protocols for clinical next-generation sequencing (NGS) need application-specific optimization. Our aim was to analyze the performance of single nucleotide variant (SNV) and copy number (CNV) detection programs on an NGS panel for a rare disease. METHODS: Thirty genes were sequenced in 83 patients with hereditary spastic paraplegia. The variant calls obtained with LifeScope, GATK UnifiedGenotyper and GATK HaplotypeCaller were compared with Sanger sequencing. The calling efficiency was evaluated for 187 (56 unique) SNVs and indels. Five multiexon deletions detected by multiple ligation probe assay were assessed from the NGS panel data with ExomeDepth, panelcn.MOPS and CNVPanelizer software. RESULTS: There were 48/51 (94%) SNVs and 1/5 (20%) indels consistently detected by all the calling algorithms. Two SNVs were not detected by any of the callers because of a rare reference allele, and one SNV in a low coverage region was only detected by two algorithms. Regarding CNVs, ExomeDepth detected 5/5 multi-exon deletions, panelcn.MOPs 4/5 and only 3/5 deletions were accurately detected by CNVPanelizer. CONCLUSIONS: The calling efficiency of NGS algorithms for SNVs is influenced by variant type and coverage. NGS protocols need to account for the presence of rare variants in the reference sequence as well as for ambiguities in indel calling. CNV detection algorithms can be used to identify large deletions from NGS panel data for diagnostic applications; however, sensitivity depends on coverage, selection of the reference set and deletion size. We recommend the incorporation of several variant callers in the NGS pipeline to maximize variant detection efficiency.

Exome sequencing is a useful diagnostic tool for complicated forms of hereditary spastic paraplegia.

Hereditary spastic paraplegias constitute a heterogeneous group of neurodegenerative diseases encompassing pure and complicated forms, for which at least 52 loci and 31 causative genes have been identified. Although mutations in the SPAST gene explain approximately 40% of the pure autosomal dominant forms, molecular diagnosis can be challenging for the sporadic and recessive forms, which are often complicated and clinically overlap with a broad number of movement disorders. The validity of exome sequencing as a routine diagnostic approach in the movement disorder clinic needs to be assessed. The main goal of this study was to explore the usefulness of an exome analysis for the diagnosis of a complicated form of spastic paraplegia. Whole-exome sequencing was performed in two Spanish siblings with a neurodegenerative syndrome including upper and lower motor neuron, ocular and cerebellar signs. Exome sequencing revealed that both patients carry a novel homozygous nonsense mutation in exon 15 of the SPG11 gene (c.2678G>A; p.W893X), which was not found in 584 Spanish control chromosomes. After many years of follow-up and multiple time-consuming genetic testing, we were able to diagnose these patients by making use of whole-exome sequencing, showing that this is a cost-efficient diagnostic tool for the movement disorder specialist.

Medical genomics: The intricate path from genetic variant identification to clinical interpretation.

The field of medical genomics involves translating high throughput genetic methods to the clinic, in order to improve diagnostic efficiency and treatment decision making. Technical questions related to sample enrichment, sequencing methodologies and variant identification and calling algorithms, still need careful investigation in order to validate the analytical step of next generation sequencing techniques for clinical applications. However, the main foreseeable challenge will be interpreting the clinical significance of the variants observed in a given patient, as well as their significance for family members and for other patients. Every step in the variant interpretation process has limitations and difficulties, and its quote of contribution to false positive and false negative results. There is no single piece of evidence enough on its own to make firm conclusions on the pathogenicity and disease causality of a given variant. A plethora of automated analysis software tools is being developed that will enhance efficiency and accuracy. However a risk of misinterpretation could derive from biased biorepository content, facilitated by annotation of variant functional consequences using previous datasets stored in the same or linked repositories. In order to improve variant interpretation and avoid an exponential accumulation of confounding noise in the medical literature, the use of terms in a standard way should be sought and requested when reporting genetic variants and their consequences. Generally, stepwise and linear interpretation processes are likely to overrate some pieces of evidence while underscoring others. Algorithms are needed that allow a multidimensional, parallel analysis of diverse lines of evidence to be carried out by expert teams for specific genes, cellular pathways or disorders.

Neurofibromatosis without Neurofibromas: Confirmation of a Genotype-Phenotype Correlation and Implications for Genetic Testing.

Neurofibromatosis type 1 (NF1) is a multisystem disease with autosomal dominant inheritance and complete penetrance diagnosed by clinical findings. Cutaneous neurofibromas are present in almost all adult patients in the dermis, epidermis or along the peripheral nerves. Plexiform neurofibromas are subcutaneous or deep lesions involving nerve plexuses or roots. Neurofibromas can degenerate into malignant tumors, with important prognostic implications. NF1 shows a broad clinic variability even within a single family. Exceptions are cases reporting the in-frame microdeletion c.2970_2972delAAT, presenting with the typical pigmentary features of NF1, but no cutaneous or plexiform neurofibromas. We report a patient with a de novo c.2970_2972delAAT mutation who had few café-au-lait spots, only 2 of which measured >15 mm, axillary and submammary freckling, a flat angioma extending over the neck, arm and trunk, a high arched palate, micrognathia, macrocephaly, pes cavus and scoliosis. There was complete absence of observable cutaneous neurofibromas as well as external plexiform neurofibromas. She had had epileptic seizures since childhood; however, a diagnosis of NF1 had not been confirmed until she was 38, partly due to the paucity of characteristic cutaneous stigmata. We confirm the association of the c.2970_2972delAAT mutation in NF1 with a particular clinical phenotype, especially with lack of detectable neurofibromas. For an appropriate management of patients and family counseling, molecular study of the NF1 gene should be considered in patients not fulfilling NIH criteria when other features suggestive of NF1 are present. In the absence of neurofibromas, starting NF1 testing with the screening of exon 17 may be worthwhile.

Asesoramiento genético en Neurología: un problema complejo que necesita regulación / Genetic counselling in Neurology: A complex problem that requires regulation

Introducción: La expansión de los estudios genéticos está transformando la práctica de la Neurología y enfrenta a los servicios clínicos con nuevos retos, como la articulación del asesoramiento genético. La amplitud de los conocimientos tanto clínicos como moleculares precisos, así como la necesidad de una evaluación psicológica y apoyo familiar, especialmente en los análisis predictivos y planificación reproductiva, hacen necesario un enfoque multidisciplinar. Desarrollo: Las características principales de las enfermedades neurodegenerativas de base genética son el elevado nivel de especialización requerido —por tratarse de enfermedades poco comunes y de difícil diagnóstico— junto con su carácter generalmente progresivo, la ausencia de tratamientos eficaces, la problemática generada por la posibilidad de estudios predictivos y la interpretación de los resultados genéticos. El objetivo del asesoramiento genético es proporcionar la información suficiente y objetiva para que cada individuo pueda tomar sus propias decisiones sobre el estudio genético. Debe incluir la evaluación de aspectos psicológicos y de comunicación familiar. El programa PICOGEN del Hospital Clinic de Barcelona para el análisis y asesoramiento genético en demencias es un buen ejemplo de una estrategia integrada capaz de abordar esta nueva situación asistencial en Neurología. Lamentablemente, este programa es una excepción en España y los pacientes con enfermedades neurogenéticas y sus familias no tienen garantizada habitualmente una asistencia adecuada.Conclusiones: El asesoramiento genético es un acto clínico per se, que precisa de un espacio, tiempo y recursos suficientes. Implica una participación multidisciplinar, atención a los aspectos psicológicos y familiares y no se puede llevar a cabo correctamente en el seno de una consulta rutinaria estándar de Neurología (AU)

Introduction: The generalization of genetic studies is transforming the practice of Neurology and confronts the clinical departments with new challenges, such as the organization of genetic counseling. The requirement of specialized knowledge, both clinical and molecular, as well as the need for psychological evaluation and family support, especially for predictive testing and reproductive planning, makes a multidisciplinary approach mandatory.Development: The main characteristics of genetic neurodegenerative diseases are the high level of required specialization — since these disorders are often rare and of difficult diagnosis— together with a generally progressive course, unavailability of effective treatment, the issues generated by predictive testing and the interpretation of genetic testing. The aim of geneticcounseling is to provide sufficient and objective information for each individual to make their own decision on genetic testing. It must touch upon psychological aspects and family communication.The PICOGEN program from the Clinic Hospital in Barcelona for genetic testing and counseling of dementias is a good example of integrated strategy capable of managing this new clinical scenario in neurology. Unfortunately, this program is an exception in Spain and the patients with neurogenetic disorders and their families usually do not have guaranteed access to an appropriate care. Conclusions: Genetic counseling is a unique clinical activity that requires provision of enough time, space and resources to be developed. It implies multidisciplinary participation, due attention to psychological and family issues, and cannot be carried out adequately in a routine Neurology clinic. Legislation is needed to promote a correct articulation of genetic counselingin our country with guarantee of quality and equity (AU)

Genetic counselling in neurology: a complex problem that requires regulation.

INTRODUCTION: The generalization of genetic studies is transforming the practice of Neurology and confronts the clinical departments with new challenges, such as the organization of genetic counseling. The requirement of specialized knowledge, both clinical and molecular, as well as the need for psychological evaluation and family support, especially for predictive testing and reproductive planning, makes a multidisciplinary approach mandatory. DEVELOPMENT: The main characteristics of genetic neurodegenerative diseases are the high level of required specialization - since these disorders are often rare and of difficult diagnosis - together with a generally progressive course, unavailability of effective treatment, the issues generated by predictive testing and the interpretation of genetic testing. The aim of genetic counseling is to provide sufficient and objective information for each individual to make their own decision on genetic testing. It must touch upon psychological aspects and family communication. The PICOGEN program from the Clinic Hospital in Barcelona for genetic testing and counseling of dementias is a good example of integrated strategy capable of managing this new clinical scenario in neurology. Unfortunately, this program is an exception in Spain and the patients with neurogenetic disorders and their families usually do not have guaranteed access to an appropriate care. CONCLUSIONS: Genetic counseling is a unique clinical activity that requires provision of enough time, space and resources to be developed. It implies multidisciplinary participation, due attention to psychological and family issues, and cannot be carried out adequately in a routine Neurology clinic. Legislation is needed to promote a correct articulation of genetic counseling in our country with guarantee of quality and equity. This includes training of the necessary health professionals, clarification of competences and provision of resources to the institutions for the development of such programs.

[Behavioral disorders in Parkinson's disease. Genetic, pharmacological and medico-legal aspects]. / Trastornos del comportamiento en la enfermedad de Parkinson. Aspectos genéticos, farmacológicos y médico-legales.

INTRODUCTION: Patients with Parkinson's disease (PD) may present neuropsychiatric and conduct disorders at different stages of the development of the disease that make treatment even more difficult. DEVELOPMENT: The neurologist must be on the lookout for the possible appearance of alterations affecting impulse control, even from the early stages of the disease, so as to be able to prevent them or to plan a suitable adjustment of treatment. Some of the most common impulsivity disorders include hypersexuality, compulsive gambling and other addictive behaviours which, if left undetected and untreated, can end up having a destructive effect on the patient's socio-familial surroundings. Psychotic disorders (hallucinations, delusions) are often associated to advanced phases of PD and to the effect of dopamine therapy, and they are associated to a higher morbidity and mortality rate. Factors of a genetic or pharmacogenetic nature or a gene-environment interaction may account for the different individual susceptibility to disorders in the neuropsychiatric realm among patients with PD. It is wise to bear in mind the possible medico-legal implications that may stem from behavioral disorders, both for the patient and his or her family and for the physician, because situations could arise that trigger conflicts between confidentiality and preventing third parties from being harmed, as well as harm that can be attributed to the side effects of medicines. CONCLUSIONS: The specialist must be familiar with, foresee and propose suitable treatment for behavioral and neuropsychiatric disorders in PD with potential medico-legal implications.

Trastornos del comportamiento en la enfermedad de Parkinson. Aspectos genéticos, farmacológicos y médico-legales / Behavioral disorders in parkinson´s disease genetic, pharmacological and medico legal aspects

Resumen. Introducción. Los pacientes con enfermedad de Parkinson (EP) pueden presentar alteraciones neuropsiquiátricas y del comportamiento en diferentes estadios evolutivos de la enfermedad que dificulten el tratamiento. Desarrollo. Es necesario que el neurólogo esté atento a la posible aparición de alteraciones en el control de los impulsos, incluso desde fases tempranas de la enfermedad, para poder prevenirlas o planificar un ajuste adecuado del tratamiento. Entre los trastornos de impulsividad más comunes están la hipersexualidad, la ludopatía y otras conductas adictivas que, si no se detectan y tratan, pueden resultar destructivas para el entorno sociofamiliar del paciente. Los trastornos de la esfera psicótica (alucinaciones, ideas delirantes) a menudo están asociados a fases avanzadas de la EP y al efecto del tratamiento dopaminérgico, y se asocian con una mayor morbimortalidad. Es posible que factores genéticos, farmacogenéticos o una interacción gen-ambiente expliquen la diferente susceptibilidad individual a alteraciones en la esfera neuropsiquiátrica entre los pacientes con EP. Es conveniente recordar las posibles implicaciones médico-legales que pueden tener los trastornos de la conducta, tanto para el paciente y su familia como para el médico, pues se podrían plantear conflictos entre la confidencialidad y la prevención de daños a terceros, así como daño atribuible al efecto secundario de medicamentos. Conclusiones. Se debe conocer, prever y tratar adecuadamente las alteraciones de la conducta y neuropsiquiátricas en la EP, con implicaciones médicas y legales (AU)

Summary. Introduction. Patients with Parkinson’s disease (PD) may present neuropsychiatric and conduct disorders at different stages of the development of the disease that make treatment even more difficult. Development. The neurologist must be on the lookout for the possible appearance of alterations affecting impulse control, even from the early stages of the disease, so as to be able to prevent them or to plan a suitable adjustment of treatment. Some of the most common impulsivity disorders include hypersexuality, compulsive gambling and other addictive behaviours which, if left undetected and untreated, can end up having a destructive effect on the patient’s socio-familial surroundings. Psychotic disorders (hallucinations, delusions) are often associated to advanced phases of PD and to the effect of dopamine therapy, and they are associated to a higher morbidity and mortality rate. Factors of a genetic or pharmacogenetic nature or a gene-environment interaction may account for the different individual susceptibility to disorders in the neuropsychiatric realm among patients with PD. It is wise to bear in mind the possible medico-legal implications that may stem from behavioral disorders, both for the patient and his or her family and for the physician, because situations could arise that trigger conflicts between confidentiality and preventing third parties from being harmed, as well as harm that can be attributed to the side effects of medicines. Conclusions. The specialist must be familiar with, foresee and propose suitable treatment for behavioral and neuropsychiatric disorders in PD with potential medico-legal implications (AU)

Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing.

The development of new methodologies for high-throughput SNP analysis is one of the most stimulating areas in genetic research. Here, we describe a rapid and robust assay to simultaneously genotype 17 mitochondrial DNA (mtDNA) coding region SNPs by minisequencing using SNaPshot. SNaPshot is a methodology based on a single base extension of an unlabeled oligonucleotide with labeled dideoxy terminators. The set of SNPs implemented in this multiplexed SNaPshot reaction allow us to allocate common mitochondrial West Eurasian haplotypes into their corresponding branch in the mtDNA skeleton, with special focus on those haplogroups lacking unambiguous diagnostic positions in the first and second hypervariable regions (HVS-I/II; by far, the most common segments analyzed by sequencing). Particularly interesting is the set of SNPs that subdivide haplogroup H; the most frequent haplogroup in Europe (40-50%) and one of the most poorly characterized phylogenetically in the HVS-I/II region. In addition, the polymorphic positions selected for this multiplex reaction increase considerably the discrimination power of current mitochondrial analysis in the forensic field and can also be used as a rapid screening tool prior to full sequencing analysis. The method has been validated in a sample of 266 individuals and shows high accuracy and robustness avoiding both the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the need for high quantities of DNA template.

[A brain tissue bank in a neuropathology laboratory. Basic methodology]. / Banco de tejidos Neurológicos en el Laboratorio de Neuropatología. Organización básica.

The Meixoeiro Hospital Brain Bank (BB) was established at the end of 2002. A BB is a tissue collection and storage system, established under the best conditions to carry out prospective morphological, biochemical or molecular studies. The BB should ideally be supported by a donor program, although samples may also be obtained from autopsy material from patients with neurodegenerative diseases. Recruitment of control cases from brains without neurological diseases is basic. The main goal of a BB is to provide brain tissue for research. Each case requires accurate clinical data, a definite diagnosis and optimal conditions of tissue preservation. The use of protocols to standardize the handling and processing of tissues, data recruitment and neuropathological diagnosis is fundamental to assure the quality and homogeneity of samples. Close collaboration between neuropathologists, neurologists and other specialists is essential in all the process. Although important advances in the tissue banking field have been achieved, the number of donors in Spain still remains low. Stronger institutional support as well as public awareness through better diffusion of the information is necessary to increase the number of donors and improve BB development.

Banco de Tejidos Neurológicos en el Laboratorio de Neuropatología. Organización básica / A Brain Bank in a Neuropathology Laboratory. Basic methodology

El Banco de Tejidos Neurológicos (BTN) del Hospital Meixoeiro se implantó a finales del año 2002. Un BTN es un sistema prospectivo de almacenamiento y conservación de tejidos en condiciones óptimas que permitan la realización de cualquier estudio morfológico, bioquímico o molecular. Su funcionamiento debe estar basado en un programa de donación, aunque las muestras pueden proceder tanto de donantes como de autopsias clínicas de pacientes con enfermedades neurodegenerativas. Es importante la obtención de cerebros de personas sin enfermedad neurológica para casos control. El objetivo principal de un BTN es el de proporcionar tejido nervioso para la investigación. Los casos deben tener una buena información clínica, un diagnóstico definitivo y unas condiciones de conservación idóneas. Para asegurar la calidad y homogeneidad de las muestras hay que estandarizar, mediante protocolos, los métodos de obtención, extracción y procesamiento del tejido, la recogida de la información y el diagnóstico neuropatológico. La colaboración multidisciplinar entre neuropatólogos, neurólogos y otros especialistas es básica en todo el proceso. Aunque se están realizando grandes avances en el campo de los Bancos Tisulares, el número de donaciones en España es aún muy bajo. Para estimular las donaciones y potenciar el desarrollo de los BTN, hay que conseguir un mayor apoyo institucional y una mayor concienciación de la sociedad mejorando la información (AU)

Análisis microgeográfico de linajes paternos y maternos en el noroeste de la península ibérica / Microgeographic patterns of paternal and maternal lineages in the North West of Iberia

Se ha analizado el efecto del muestreo y la subestructuración poblacional en la estima de frecuencias haplotípicas de cromosoma Y y ADN mitocondrial en las poblaciones de Galicia y Cantabria (NO España).En Galicia se encontraron diferencias significativas en las frecuencias de haplogrupos de SNPs entre una muestra de población general y otra de pequeños núcleos rurales, observándose sin embargo una gran homogeneidad tanto en linajes maternos como paternos. Por el contrario, la población cántabra presenta una marcada subestructura genética tanto en el cromosoma Y como en el ADNmt. En este trabajo analizamos las implicaciones forenses y antropológicas de estos resultados (AU)

The use of the LightCycler for the detection of Y chromosome SNPs.

A novel methodology based on PCR monitoring on-line with fluorescent formats using the LightCycler for Y chromosome SNP typing is proposed. The main advantages of the system are the time necessary for the analysis (which is around 20 min), the robustness and the accuracy of the method and especially its sensitivity, which permits the detection of the male component in male-female mixtures up to 1:300 for some of the SNPs. Singleplexes of four different SNPs (M9, sY81, SRY-1532 and SRY-2627) as well as two duplexes (M9 and sY81 on the one hand and SRY-1532 and SRY-2627 on the other) were efficiently implemented. A simultaneous amplification and analysis of the four SNPs is also possible. It seems difficult with the current methodology to implement more than a quadruplex.