Investigating the correlation between genotype and phenotype in Prader-Willi syndrome: a study of 45 cases from Brazil.

BACKGROUND: Prader-Willi syndrome (PWS) is a genetic disorder characterized by abnormalities in the 15q11-q13 region. Understanding the correlation between genotype and phenotype in PWS is crucial for improved genetic counseling and prognosis. In this study, we aimed to investigate the correlation between genotype and phenotype in 45 PWS patients who previously underwent methylation-sensitive high-resolution melting (MS-HRM) for diagnosis. RESULTS: We employed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and Sanger sequencing, along with collecting phenotypic data from the patients for comparison. Among the 45 patients, 29 (64%) exhibited a deletion of 15q11-q13, while the remaining 16 (36%) had uniparental disomy. No statistically significant differences were found in the main signs and symptoms of PWS. However, three clinical features showed significant differences between the groups. Deletion patients had a higher prevalence of myopia than those with uniparental disomy, as well as obstructive sleep apnea and an unusual skill with puzzles. CONCLUSIONS: The diagnostic tests (MS-HRM, MS-MLPA, and Sanger sequencing) yielded positive results, supporting their applicability in PWS diagnosis. The study's findings indicate a general similarity in the genotype-phenotype correlation across genetic subtypes of PWS.

Establishing a Standardized DNA Extraction Method Using NaCl from Oral Mucosa Cells for Its Application in Imprinting Diseases Such as Prader-Willi and Angelman Syndromes: A Preliminary Investigation.

BACKGROUND: Diagnosing imprinting defects in neonates and young children presents challenges, often necessitating molecular analysis for a conclusive diagnosis. The isolation of genetic material from oral swabs becomes crucial, especially in settings where blood sample collection is impractical or for vulnerable populations like newborns, who possess limited blood volumes and are often too fragile for invasive procedures. Oral swab samples emerge as an excellent source of DNA, effectively overcoming obstacles associated with rare diseases. METHODS: In our study, we specifically addressed the determination of the quality and quantity of DNA extracted from oral swab samples using NaCl procedures. RESULTS: We compared these results with extractions performed using a commercial kit. Subsequently, the obtained material underwent MS-HRM analysis for loci associated with imprinting diseases such as Prader-Willi and Angelman syndromes. CONCLUSIONS: Our study emphasizes the significance of oral swab samples as a reliable source for obtaining DNA for MS-HRM analysis. NaCl extraction stands out as a practical and cost-effective method for genetic studies, contributing to a molecular diagnosis that proves particularly beneficial for patients facing delays in characterization, ultimately influencing their treatment.

A Comprehensive Review of Syndromic Forms of Obesity: Genetic Etiology, Clinical Features and Molecular Diagnosis.

Syndromic obesity refers to obesity occurring with additional clinical findings, such as intellectual disability/developmental delay, dysmorphic features, and congenital malformations. PURPOSE OF REVIEW: To present a narrative review regarding the genetic etiology, clinical description, and molecular diagnosis of syndromic obesity, which is a rare condition with high phenotypic variability and genetic heterogeneity. The following syndromes are presented in this review: Prader-Willi, Bardet-Biedl, Pseudohypoparathyroidism, Alström, Smith-Magenis, Cohen, Temple, 1p36 deletion, 16p11.2 microdeletion, Kleefstra, SIM1-related, Börjeson-Forssman-Lehmann, WAGRO, Carpenter, MORM, and MYT1L-related syndromes. RECENT FINDINGS: There are three main groups of mechanisms for syndromic obesity: imprinting, transcriptional activity regulation, and cellular cilia function. For molecular diagnostic, methods of genome-wide investigation should be prioritized over sequencing of panels of syndromic obesity genes. In addition, we present novel syndromic conditions that need further delineation, but evidences suggest they have a higher frequency of obesity. The etiology of syndromic obesity tends to be linked to disrupted neurodevelopment (central) and is associated with a diversity of genes and biological pathways. In the genetic investigation of individuals with syndromic obesity, the possibility that the etiology of the syndromic condition is independent of obesity should be considered. The accurate genetic diagnosis impacts medical management, treatment, and prognosis, and allows proper genetic counseling.

[Epigenetics of Prader-Willi Syndrome: A good example of translational medicine]. / Epigenética del síndrome de Prader-Willi: un buen ejemplo de medicina traslacional.

In this editorial the author presents a study, concerning Prader-Willi syndrome, which is paradigmatic for translational medicine, given that it creates a synergy between genetics and molecular biology, in order to improve the care for patients suffering from this syndrome.

En el presente editorial el autor expone un estudio, relacionado con el síndrome de Prader-Willi, que es paradigmático para la medicina traslacional, dado que crea una sinergia entre la genética y la biología molecular, a fin de mejorar la atención a los pacientes que padecen este síndrome.

[Prader-Willi and Angelman syndromes: case series diagnosed by MS-MLPA assay]. / Síndromes Prader-Willi y Angelman: serie de casos diagnosticados mediante la técnica de MS-MLPA.

BACKGROUND: Prader Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders caused by deletions or methylation defects, making a loss of expression of imprinted genes located in the 15q11-q13 region, and these can be assessed by different cytogenomic and molecular techniques. We report a case series of patients with PWS and AS evaluated through the MS-MLPA assay. CLINICAL CASES: We studied four patients with a clinical diagnosis of PWS and another with AS, evaluated as far as possible with karyotype and FISH, and with MS-MLPA assay for the 15q11-q13 region in all cases. In patients with PWS, neonatal hypotonia was the main reason for consultation and in three of them we identified a deletion of 15q11-q13 by MS-MLPA, also confirmed by FISH; and in the other one, an abnormal methylation pattern consistent with a maternal uniparental disomy. The patient with AS presented with a typical picture which led to the identification of a deletion in 15q11-q13 by MS-MLPA, also confirmed by FISH. CONCLUSIONS: The use of the MS-MLPA assay for the 15q11-q13 region was very useful for the diagnosis and identification of the genomic and epigenetic defects involved in either PWS and AS.

INTRODUCCIÓN: el síndrome Prader-Willi (SPW) y el síndrome de Angelman (SA) son trastornos del neurodesarrollo producidos por deleciones o defectos de metilación que producen pérdida de expresión en los genes improntados de la región 15q11 q13, mismos que pueden ser evaluados por diferentes técnicas citogenómicas y moleculares. Presentamos una serie de pacientes con SPW y SA en los que se identificó el tipo de defecto de la región 15q11-q13 mediante la técnica de MS-MLPA. CASOS CLÍNICOS: estudiamos cuatro pacientes con diagnóstico clínico de SPW y uno con SA, evaluados en lo posible con cariotipo, FISH y todos con ensayo MS-MLPA para la región 15q11-q13. En los pacientes con SPW, la hipotonía neonatal fue el motivo principal de consulta. En tres de ellos se identificó deleción de 15q11-q13 por MS-MLPA, confirmada por FISH, y en uno el patrón de metilación anormal fue compatible con una disomía uniparental materna. El paciente con SA presentó un cuadró típico y también se identificó una deleción en 15q11-q13 por MS-MLPA, confirmada por FISH. CONCLUSIONES: confirmamos que el uso de la técnica de MS-MLPA para la región 15q11 q13 mostró ser de gran utilidad para identificar los mecanismos genómicos y epigenéticos implicados en el SPW y el SA.

Correlação entre o genótipo e o fenótipo dos pacientes com a Síndrome de Prader-Willi / Correlation between the genotype and phenotype of patients with Prader-Willi Syndrome

A síndrome de Prader-Willi (SPW) é uma doença multissistêmica, cujas manifestações principais incluem hipotonia, obesidade, leve atraso mental, hipogonadismo e insuficiência do hormônio de crescimento. A SPW foi a primeira desordem genética descrita envolvida com o imprinting genômico. O imprinting genômico é uma modificação epigenética do DNA responsável por metilar as ilhas CpG, presentes em regiões promotoras dos genes, inativando a expressão deste gene. Na SPW, o indivíduo possui o alelo materno quimicamente inativado através do imprinting, além disso, o indivíduo perde a função dos mesmos genes no alelo paterno devido a 3 possíveis mecanismos genéticos: Deleção, dissomia uniparental materna (DUM), e microdeleções ou defeitos no centro de controle do imprinting. Ainda há muito a se entender sobre as bases genéticas da SPW e sua correlação com os fenótipos clínicos vistos nestes pacientes, e esta comparação entre o perfil molecular e os sintomas clínicos vistos em pacientes com a SPW vem sendo um tema muito discutido dentro da literatura. Muitos achados suportam uma possível correlação entre o genótipo e o fenótipo destes pacientes. Estabelecendo uma possível correlação entre genótipo e fenótipo irá trazer uma maior compreensão da SPW, provendo um melhor aconselhamento genético e consequentemente melhorando o prognóstico para estes indivíduos e suas famílias. Este estudo tem como objetivo identificar a associação dos diferentes mecanismos genéticos da SPW com os diversos sintomas clínicos, contribuindo para um melhor prognóstico da doença. Um estudo descritivo de pesquisa básica e quantitativa a partir de amostras de sangue periférico de 45 pacientes com padrão de metilação compatível com a SPW acompanhados no Centro de Genética Médica do IFF/FIOCRUZ e pelo Instituto Estadual de Diabetes e Endocrinologia do Estado do rio de Janeiro (IEDE/RJ). O estudo vemsendo desenvolvido no Laboratório de Alta Complexidade do IFF (LACIFF). A abordagem metodológica consistiu no rastreamento destes 45 pacientes utilizando a técnica de MS HRM; MS-MLPA visando identificar as deleções nos pacientes; e o sequenciamento de Sanger visando identificar as dissomias uniparentais maternas e os defeitos no centro de controle do imprinting. Posteriormente foi realizado a coleta de dados fenotípicos dos pacientes que apresentaram alterações compatíveis com a SPW. O trabalho em questão tem como resultados esperados encontrar uma correlação genótipo ­ fenótipo, visando um melhor entendimento sobre as bases genéticas da síndrome e um melhor prognóstico para estes pacientes e suas famílias.

Prader-Willi syndrome (PWS) is a multisystemic disease, the main manifestations of which include hypotonia, obesity, mild mental retardation, hypogonadism, and insufficient growth hormone. SPW was the first described genetic disorder involved with genomic imprinting. Genomic imprinting is an epigenetic modification of the DNA responsible for the methylation of the CpG islands, present in promoter regions of the genes, inactivating the expression of this gene. In PWS, the individual has the maternally allele chemically inactivated through imprinting, in addition, the individual loses the function of the same genes in the paternal allele due to 3 possible genetic mechanisms: Deletion, maternal uniparental disomy (matUPD), and micro deletions or defects in the imprinting control center. There is still a lot to understand about the genetic bases of PWS and its correlation with the clinical phenotypes seen in these patients, and this parallel between the molecular profile and the clinical symptoms seen in patients with PWS has been a very discussed topic in the literature. Many findings support a possible correlation between the genotype and phenotype of these patients. Establishing a possible correlation between genotype and phenotype, will bring a greater understanding of PWS, providing better genetic counseling and consequently improving the prognosis for these individuals and their families. This study aims to identify the association of the different genetic mechanisms of PWS with the different clinical symptoms, contributing to a better prognosis of the disease. A descriptive study of basic and quantitative research based on peripheral blood samples from 45 patients with methylation status compatible with PWS followed at the Medical Genetics Center of IFF / FIOCRUZ and by the State Institute of Diabetes and Endocrinology of the State of Rio de Janeiro (IEDE / RJ). The study has been developed at the IFF High Complexity Laboratory (LACIFF). The methodological approach consisted of tracking these 45 patients using the MS-HRM technique; MS MLPA to identify deletions in patients; and the Sanger sequencing aiming to identify maternal uniparental dissomies and defects in the imprinting control center. Subsequently, phenotypic data were collected from patients who presented changes compatible with PWS. The work in question has as expected results to find this genotype - phenotype correlation, aiming at a better understanding about the genetic bases of the syndrome and a better prognosis for these patients and their families.

A newborn screening pilot study using methylation-sensitive high resolution melting on dried blood spots to detect Prader-Willi and Angelman syndromes.

Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct imprinted disorders characterized by genetic abnormalities at 15q11-q13. Early diagnosis of both syndromes provides improved treatment and accurate genetic counseling. Whole blood (WB) is the most common DNA source of many methodologies to detect PWS and AS, however, the need of WB makes a massive screening difficult in newborns due to economic and technical limitations. The aim of this study was to adapt a Methylation-sensitive High-Resolution Melting (MS-HRM) approach from dried blood spot (DBS) samples, assessing the different DNA isolation techniques and diagnostic performance. Over a 1-year period, we collected 125 DBS cards, of which 45 had already been diagnosed by MS-HRM (20 PWS, 1 AS, and 24 healthy individuals). We tested three different DBS-DNA extraction techniques assessing the DNA concentration and quality, followed by MS-HRM and statistical comparison. Each DBS-DNA extraction method was capable of accuracy in detecting all PWS and AS individuals. However, the efficiency to detect healthy individuals varied according to methodology. In our experience, DNA extracted from DBS analyzed by the MS-HRM methodology provides an accurate approach for genetic screening of imprinting related disorders in newborns, offering several benefits compared to traditional whole blood methods.

Prader-Willi syndrome: endocrine manifestations and management.

Prader-Willi syndrome (PWS) is a genetic disorder caused by the absence of gene expression in the 15q11.2-q13 paternal chromosome. Patients with PWS develop hypothalamic dysfunction that can lead to various endocrine changes such as: obesity, growth hormone deficiency, hypogonadism, hypothyroidism, adrenal insufficiency and low bone mineral density. In addition, individuals with PWS have increased risk of developing type 2 diabetes mellitus. This review summarizes and updates the current knowledge about the prevention, diagnosis and treatment of endocrine manifestations associated with Prader Willi syndrome, especially diagnosis of growth hormone deficiency, management and monitoring of adverse effects; diagnosis of central adrenal insufficiency and management in stressful situations; screening for central hypothyroidism; research and treatment of hypogonadism; prevention and treatment of disorders of glucose metabolism. Careful attention to the endocrine aspects of PWS contributes significantly to the health of these individuals. Arch Endocrinol Metab. 2020;64(3):223-34.

Prader-Willi syndrome: endocrine manifestations and management

ABSTRACT Prader-Willi syndrome (PWS) is a genetic disorder caused by the absence of gene expression in the 15q11.2-q13 paternal chromosome. Patients with PWS develop hypothalamic dysfunction that can lead to various endocrine changes such as: obesity, growth hormone deficiency, hypogonadism, hypothyroidism, adrenal insufficiency and low bone mineral density. In addition, individuals with PWS have increased risk of developing type 2 diabetes mellitus. This review summarizes and updates the current knowledge about the prevention, diagnosis and treatment of endocrine manifestations associated with Prader Willi syndrome, especially diagnosis of growth hormone deficiency, management and monitoring of adverse effects; diagnosis of central adrenal insufficiency and management in stressful situations; screening for central hypothyroidism; research and treatment of hypogonadism; prevention and treatment of disorders of glucose metabolism. Careful attention to the endocrine aspects of PWS contributes significantly to the health of these individuals. Arch Endocrinol Metab. 2020;64(3):223-34

Case of 15q26-qter deletion associated with a Prader-Willi phenotype.

Prader-Willi syndrome (PWS) is one of the common neurogenetic disorders associated with intellectual disability. PWS involves a complex inheritance pattern and is caused by an absence of gene expression on the paternally inherited 15q11.2-q13 region, either due to deletion, maternal uniparental disomy or imprinting defect. The syndrome is characterized principally by severe neonatal hypotonia, a weak suck in infancy that is later followed by hyperphagia and obesity, developmental delay, intellectual disability and short stature. In the case of the chromosome 15q26-qter deletion syndrome or Drayer's syndrome, very few reports have been published. Its characteristics include intrauterine growth restriction, postnatal growth failure, varying degrees of intellectual disability, developmental delay, typical facial appearance and diaphragmatic hernia. The present paper describes a female patient in whom clinical findings were suggestive of PWS and deletion in the 15q26-qter region. Both karyotyping and methylation-specific polymerase chain reaction were shown to be normal. Nevertheless, fluorescence in situ hybridization showed a 15qter deletion that was later mapped by single nucleotide polymorphism (SNP)-array. The deleted genomic region involves the insulin-like growth factor-1 receptor (IGF1R) gene, which is related to short stature, developmental delay and intellectual disability. This case had various clinical characteristics in common with the cases of 15q26-qter deletionand characteristics compatible with PWS.

A rapid and accurate methylation-sensitive high-resolution melting analysis assay for the diagnosis of Prader Willi and Angelman patients.

BACKGROUND: Prader Willi (PWS) and Angelman (AS) syndromes are rare genetic disorders characterized by deletions, uniparental disomy, and imprinting defects at chromosome 15. The loss of function of specific genes caused by genetic alterations in paternal allele causes PWS while the absence in maternal allele results AS. The laboratory diagnosis of PWS and AS is complex and demands molecular biology and cytogenetics techniques to identify the genetic mechanism related to the development of the disease. The DNA methylation analysis in chromosome 15 at the SNURF-SNRPN locus through MS-PCR confirms the diagnosis and distinguishes between PWS and AS. Our study aimed to establish the MS-PCR technique associated with High-Resolution Melting (MS-HRM) in PWS and AS diagnostic with a single pair of primers. METHODS: We collected blood samples from 43 suspected patients to a cytogenetic and methylation analysis. The extracted DNA was treated with bisulfite to perform comparative methylation analysis. RESULTS: MS-HRM and MS-PCR agreed in 100% of cases, identifying 19(44%) PWS, 3(7%) AS, and 21(49%) Normal. FISH analysis detected four cases of PWS caused by deletions in chromosome 15. CONCLUSION: The MS-HRM showed good performance with a unique pair of primers, dispensing electrophoresis gel analysis, offering a quick and reproducible diagnostic.

Aplicação do sequenciamento de nova geração no diagnóstico molecular da síndrome de Prader-Willi / Application of next-generation sequencing in the molecular diagnosis of Prader-Willi syndrome

Introdução: A síndrome de Prader- Willi (SPW) é uma desordem genética complexa, caracterizada por deleções, dissomia uniparental materna ou defeito no centro de imprinting no alelo paterno do cromossomo 15. As perdas de funções de genes específicos da região 15q11 afetam múltiplos sistemas corporais. O diagnóstico da SPW envolve a realização de diversas técnicas de biologia molecular e citogenética para a completa elucidação do mecanismo genético relacionado ao desenvolvimento da síndrome, tornando todo o processo laborioso, demorado e custoso. Objetivo: Aplicação da tecnologia sequenciamento de nova geração (NGS), plataforma Ion Torrent PGM, para diagnostico molecular da SPW. Materiais e Métodos: Foram incluídos 17 pacientes suspeitos para SPW onde foram submetidos a análise de metilação (MS-HRM), citogenética (GTG e FISH) e sequenciamento de genes alvos na plataforma Ion Torrent PGM, e subsequente confirmação de variantes através da técnica de sequenciamento de Sanger. Resultados: A análise de metilação detectou 6 indivíduos portadores da SPW, 1 portador da Síndrome de Angelman (SA) e 10 indivíduos normais. A técnica de GTG identificou 1 indivíduo com uma grande perda cromossômica, já a metodologia de FISH identificou 3 indivíduos com deleções, totalizando 4. O bom desempenho da tecnologia de sequenciamento NGS, através da metodologia Ion Torrent PGM, permitiu a realização de análises de frequência alélica, variação do número de cópias (CNV) e de mutações específicas do genoma. As análises por bioinformática realizadas nos dados do NGS permitiram detectar 4 pacientes portadores de deleção, classificando-as como Tipo 1 ou Tipo 2. Além disso, foi possível identificar um evento raro de dissomia uniparental segmentar, com complicações prognósticas severas. Identificou-se variantes do tipo INDEL no gene PWRN1 em 2 pacientes, onde, o impacto funcional desta mutação ainda não foi estudado. Contudo o gene possui forte correlação com o desenvolvimento da SPW. A metodologia também identificou uma mutação INDEL no gene MAGEL2 em um paciente com padrão de metilação normal no MS-HRM, sugerindo a identificação de uma síndrome análoga a SPW. Todas as variantes detectadas foram validadas através do sequenciamento de Sanger. Conclusão: A plataforma Ion Torrent identificadou todas as alterações relacionadas ao desenvolvimento da SPW. O pipeline desenvolvido mostrou-se aplicável a uma rotina diagnóstica.

Introduction: Prader-Willi syndrome (PWS) is a complex genetic disorder characterized by deletions, maternal uniparental disomy or defect at the imprinting center in the paternal allele of chromosome 15. Loss of 15q11 region-specific gene functions affects multiple body systems. The diagnosis of SPW involves the accomplishment of several techniques of molecular biology and cytogenetics for the complete elucidation of the genetic mechanism related to the development of the syndrome, making the whole process laborious, time-consuming and costly. Objective: Application of the Next-Generation sequencing technology (NGS), platform Ion Torrent PGM, for molecular diagnosis of PWS. Materials and Methods: Seventeen suspected patients for SPW were submitted to methylation (MS-HRM), cytogenetic analysis (GTG and FISH) and sequencing of target genes in the Ion Torrent PGM platform, and subsequent confirmation of variants by the technique of sequencing of Sanger. Results: Methylation analysis detected 6 individuals with SPW, 1 with Angelman Syndrome (AS) and 10 normal individuals. The GTG technique identified 1 individual with a large chromosomal loss, and the FISH methodology identified 3 individuals with deletions, totaling 4. The good performance of the NGS sequencing technology, through the Ion Torrent PGM methodology, allowed the performance of frequency analyzes allelic, copy number variation (CNV), and genome-specific mutations. The bioinformatics analyses performed on the NGS data allowed the detection of 4 patients with deletion, classifying them as Type 1 or Type 2. In addition, it was possible to identify a rare segmental uniparental disomy with severe prognostic complications. Variables of the INDEL type were identified in the PWRN1 gene in 2 patients, where the functional impact of this mutation has not been studied. However, the gene has a strong correlation with the development of PWS. The methodology also identified an INDEL mutation in the MAGEL2 gene in a patient with normal methylation pattern in MS-HRM, suggesting the identification of a syndrome similar to PWS. All detected variants were validated through Sanger sequencing. Conclusion: The Ion Torrent platform has identified all the changes related to the development of PWS. The pipeline developed proved to be applicable to a diagnostic routine.

Análisis de variación del número de copias y de patrones de metilación en la región 15q11-q13 / Variation analysis of the number of copies and methylene patterns in region 15q11-q13

La región q11-q13 del cromosoma 15 humano es proclive a sufrir alteraciones genéticas. Algunos genes de la región presentan expresión parental diferencial monoalélica, regulada por imprinting (EI). Errores en la regulación del EI, disomías uniparentales (DSU), así como también el cambio en el número de copias genómicas (CNV) producidos por sitios susceptibles de quiebre cromosómico (BP), producen alteraciones en esta región. Las enfermedades más frecuentes asociadas son el síndrome de Prader-Willi, el síndrome de Angelman y el síndrome de microduplicación 15q11-q13. En el presente trabajo analizamos la región 15q11-q13 por Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA) en 181 muestras de ADN derivadas a nuestro servicio de análisis genético molecular. En este trabajo mostramos que, de las 181 muestras, 39 presentaron alteraciones detectables por MS-MLPA. El 61.5% (24/39) de esas alteraciones detectadas fueron deleciones, el 5.1% (2/39) duplicaciones y el 33.3%(13/39) DSU/EI. Los CNV fueron 4 veces más frecuentes que las DSU/EI (OR = 4; IC 95%: 1.56-10.25) consistente con la literatura. Entre los CNV, dos casos atípicos permiten postular posibles sitios BP que no han sido informados en la literatura previamente.

Human chromosome 15q11-q13 region is prone to suffer genetic alterations. Some genes of this region have a differential monoallelic imprinting-regulated expression pattern. Defects in imprinting regulation (IE), uniparental disomy (UPD) or copy number variation (CNV) due to chromosomal breakpoints (BP) in 15q11-q13 region, are associated with several diseases. The most frequent are Prader-Willi syndrome, Angelman syndrome and 15q11-q13 microduplication syndrome. In this work, we analyzed DNA samples from 181 patients with phenotypes which were compatible with the above-mentioned diseases, using Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA). We show that, of the 181 samples, 39 presented alterations detectable by MS-MLPA. Of those alterations, 61.5% (24/39) were deletions, 5.1% (2/39) duplications and 33.3% (13/39) UPD/IE. The CNV cases were 4 times more frequent than UPD/IE (OR= 4; IC 95%: 1.56-10.25), consistent with the literature. Among the CNVs, two atypical cases allow to postulate new possible BP sites that have not been reported previously in the literature.

[Variation analysis of the number of copies and methylene patterns in region 15q11-q13]. / Análisis de variación del número de copias y de patrones de metilación en la región 15q11-q13.

Human chromosome 15q11-q13 region is prone to suffer genetic alterations. Some genes of this region have a differential monoallelic imprinting-regulated expression pattern. Defects in imprinting regulation (IE), uniparental disomy (UPD) or copy number variation (CNV) due to chromosomal breakpoints (BP) in 15q11-q13 region, are associated with several diseases. The most frequent are Prader-Willi syndrome, Angelman syndrome and 15q11-q13 microduplication syndrome. In this work, we analyzed DNA samples from 181 patients with phenotypes which were compatible with the above-mentioned diseases, using Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA). We show that, of the 181 samples, 39 presented alterations detectable by MS-MLPA. Of those alterations, 61.5% (24/39) were deletions, 5.1% (2/39) duplications and 33.3% (13/39) UPD/IE. The CNV cases were 4 times more frequent than UPD/IE (OR= 4; IC 95%: 1.56-10.25), consistent with the literature. Among the CNVs, two atypical cases allow to postulate new possible BP sites that have not been reported previously in the literature.

Dual molecular diagnosis contributes to atypical Prader-Willi phenotype in monozygotic twins.

We describe monozygotic twin girls with genetic variation at two separate loci resulting in a blended phenotype of Prader-Willi syndrome and Pitt-Hopkins syndrome. These girls were diagnosed in early infancy with Prader-Willi syndrome, but developed an atypical phenotype, with apparent intellectual deficiency and lack of obesity. Array-comparative genomic hybridization confirmed a de novo paternal deletion of the 15q11.2q13 region and exome sequencing identified a second mutational event in both girls, which was a novel variant c.145+1G>A affecting a TCF4 canonical splicing site inherited from the mosaic mother. RNA studies showed that the variant abolished the donor splicing site, which was accompanied by activation of an alternative non-canonical splicing-site which then predicts a premature stop codon in the following exon. Clinical re-evaluation of the twins indicated that both variants are likely contributing to the more severe phenotypic presentation. Our data show that atypical clinical presentations may actually be the expression of blended clinical phenotypes arising from independent pathogenic events at two loci.

A boy with Prader-Willi syndrome: unmasking precocious puberty during growth hormone replacement therapy.

Prader-Willi syndrome (PWS) is a genetic disorder frequently characterized by obesity, growth hormone deficiency, genital abnormalities, and hypogonadotropic hypogonadism. Incomplete or delayed pubertal development as well as premature adrenarche are usually found in PWS, whereas central precocious puberty (CPP) is very rare. This study aimed to report the clinical and biochemical follow-up of a PWS boy with CPP and to discuss the management of pubertal growth. By the age of 6, he had obesity, short stature, and many clinical criteria of PWS diagnosis, which was confirmed by DNA methylation test. Therapy with recombinant human growth hormone (rhGH) replacement (0.15 IU/kg/day) was started. Later, he presented psychomotor agitation, aggressive behavior, and increased testicular volume. Laboratory analyses were consistent with the diagnosis of CPP (gonadorelin-stimulated LH peak 15.8 IU/L, testosterone 54.7 ng/dL). The patient was then treated with gonadotropin-releasing hormone analog (GnRHa). Hypothalamic dysfunctions have been implicated in hormonal disturbances related to pubertal development, but no morphologic abnormalities were detected in the present case. Additional methylation analysis (MS-MLPA) of the chromosome 15q11 locus confirmed PWS diagnosis. We presented the fifth case of CPP in a genetically-confirmed PWS male. Combined therapy with GnRHa and rhGH may be beneficial in this rare condition of precocious pubertal development in PWS.

A boy with Prader-Willi syndrome: unmasking precocious puberty during growth hormone replacement therapy

SUMMARY Prader-Willi syndrome (PWS) is a genetic disorder frequently characterized by obesity, growth hormone deficiency, genital abnormalities, and hypogonadotropic hypogonadism. Incomplete or delayed pubertal development as well as premature adrenarche are usually found in PWS, whereas central precocious puberty (CPP) is very rare. This study aimed to report the clinical and biochemical follow-up of a PWS boy with CPP and to discuss the management of pubertal growth. By the age of 6, he had obesity, short stature, and many clinical criteria of PWS diagnosis, which was confirmed by DNA methylation test. Therapy with recombinant human growth hormone (rhGH) replacement (0.15 IU/kg/day) was started. Later, he presented psychomotor agitation, aggressive behavior, and increased testicular volume. Laboratory analyses were consistent with the diagnosis of CPP (gonadorelin-stimulated LH peak 15.8 IU/L, testosterone 54.7 ng/dL). The patient was then treated with gonadotropin-releasing hormone analog (GnRHa). Hypothalamic dysfunctions have been implicated in hormonal disturbances related to pubertal development, but no morphologic abnormalities were detected in the present case. Additional methylation analysis (MS-MLPA) of the chromosome 15q11 locus confirmed PWS diagnosis. We presented the fifth case of CPP in a genetically-confirmed PWS male. Combined therapy with GnRHa and rhGH may be beneficial in this rare condition of precocious pubertal development in PWS.

A modified MS-PCR approach to diagnose patients with Prader-Willi and Angelman syndrome.

Prader-Willi (PWS) and Angelman (AS) syndromes are clinically distinct neurodevelopmental genetic diseases with multiple phenotypic manifestations. They are one of the most common genetic syndromes caused by non-Mendelian inheritance in the form of genomic imprinting, and can be attributable to the loss of gene expression due to imprinting within the chromosomal region 15q11-q13. Clinical diagnosis of PWS and AS is challenging, and the use of molecular and cytomolecular studies is recommended to help in determining the diagnosis of these conditions. The methylation analysis is a sensible approach; however there are several techniques for this purpose, such as the methylation-sensitive polymerase chain reaction (MS-PCR). This study aims to optimize the MS-PCR assay for the diagnosis of potential PWS and AS patients using DNA modified by sodium bisulfite. We used the MS-PCR technique of PCR described by Kosaki et al. (1997) adapted with betaine. All different concentrations of betaine used to amplify the methylated and unmethylated chromosomal region 15q11-q13 on the gene SNRPN showed amplification results, which increased proportionally to the concentration of betaine. The methylation analysis is a technically robust and reproducible screening method for PWS and AS. The MS-PCR assures a faster, cheaper and more efficient method for the primary diagnosis of the SNRPN gene in cases with PWS and AS, and may detect all of the three associated genetic abnormalities: deletion, uniparental disomy or imprinting errors.

Caracterización clínico genética del síndrome Prader Willi / Clinic genetic characterization of Prader-Willi syndrome

El síndrome Prader Willi es un desorden genético causado por la pérdida de genes contenidos en la región 15q11-q13 del cromosoma paterno.Describir las características clínicas y genéticas de los pacientes con síndrome Prader Willi.Se realizó un estudio descriptivo, de corte transversal, con el universo de 15 pacientes con sospecha de síndrome Prader Willi remitidos a consulta provincial de Genética Clínica durante el año 2013. Se consideraron como variables clínicas los criterios diagnósticos según Holms, y como variables genéticas los resultados de los estudios cromosómicos y moleculares.Predominó el sexo femenino en un 66.7 por ciento. Las edades estuvieron entre los tres y los 41 años. Los criterios mayores más frecuentes resultaron la obesidad troncular y el retraso del neurodesarrollo en el 100 por ciento de los pacientes. Los criterios menores más identificados fueron los disturbios del sueño y las dificultades del lenguaje con un 66.7 por ciento cada uno. En ninguno de los casos se detectaron anomalías cromosómicas por cariotipificación. Tres pacientes (60 por ciento) presentaron la deleción a nivel de la región 15q11-q13 identificada por la técnica de hibridación in sito con fluorescencia.La definición del diagnóstico en la provincia resulta demorada. Se requiere de reevaluación según los criterios clínicos en las diferentes etapas de la vida para diagnóstico de certeza. La presencia de hipotonía neonatal y dificultades en la alimentación son elementos asociados al diagnóstico por deleción 15q11-q13(AU)

Prader-Willi syndrome is a genetic disorder caused by deleted or unexpressed genes contained in 15q11-q13 region of paternal chromosome. The objective was to describe clinical and genetic characteristics of patients with Prader-Willi syndrome.A descriptive, cross-sectional study was conducted in 15 patients with Prader-Willi suspect, who were referred to the provincial office of Clinical Genetics during 2013. As clinical variables the diagnostic criteria of Holms were considered and as genetic variables, the results of chromosomal and molecular studies.Female sex prevailed in 66.7 per cent. Ages were between 3 and 41. Most frequent major criteria were troncular obesity and neurodevelopment retardation in 100 por ciento of the patients. The minor criteria more identified were: sleep disturbances and speech difficulties (66.7 per cent each one). None of the cases presented chromosomal anomalies because of karyotype classification. Three patients (60 per cent) presented deletion at 15q11-q13 region level, which was identified by hybridization in situ with fluorescence.In the province a definitive diagnosis was delayed. A reassessment is required according to the clinical criteria during the different stages of life in order to achieve a definitive diagnosis. The presence of neonatal hypotonia and difficulties in feeding are associated elements to the diagnosis of 15q11-q13 deletion(AU)