Prenatal diagnosis of a fetus with Phelan-McDermid syndrome and 21q21 microdeletion by multiple genetic techniques / 中华医学遗传学杂志

OBJECTIVE@#To carry out prenatal diagnose for a fetus with ultrasonography abnormalities using multiple genetic techniques.@*METHODS@#Routine G-banding chromosomal analysis and single nucleotide polymorphism array (SNP-array) were applied in conjunction for the prenatal diagnosis of the fetus. The result was confirmed by fluorescence in situ hybridization (FISH).@*RESULTS@#SNP-array detected that the fetus has carried a hemizygous 5.1 Mb deletion at 22q13.31q13.33, which is associated with Phelan-McDermid syndrome, and a hemizygous 4.5 Mb deletion at 21q21.1q21.2. FISH analysis of the fetus and its parents suggested that both deletions were de novo in origin.@*CONCLUSION@#The hemizygous deletions on 21q21.1q21.2 and 22q13.31q13.33 probably underlay the abnormal phenotype of the fetus. Genetic analysis can provide crucial information for the prenatal diagnosis and genetic counseling.

Gene variant analysis of a child presented with neonatal diabetes and multiple organ malformations / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for an infant with neonatal diabetes (NDM) and multiple malformations.@*METHODS@#Genetic variants were detected by next generation sequencing (NGS). Suspected variant was verified by Sanger sequencing.@*RESULTS@#A de novo heterozygous variant, c.1454_1455del(p.K485Rfs), was detected in exon 5 of the GATA6 gene. The variant was undetected in his parents and unreported previously. Bioinformatic analysis predicted the variant to be pathogenic.@*CONCLUSION@#The heterozygous variant of c.1454_1455del(p.K485Rfs) of the GATA6 gene probably underlies the disease in this child. Genetic testing can facilitate diagnosis and genetic counseling for NDM.

Arboviral diseases in the Western Brazilian Amazon: a perspective and analysis from a tertiary health & research center in Manaus, State of Amazonas

The Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), located in Manaus, the capital of the State of Amazonas (Western Brazilian Amazon), is a pioneering institution in this region regarding the syndromic surveillance of acute febrile illness, including arboviral infections. Based on the data from patients at the FMT-HVD, we have detected recurrent outbreaks in Manaus by the four dengue serotypes in the past 15 years, with increasing severity of the disease. This endemicity has culminated in the simultaneous circulation of all four serotypes in 2011, the first time this has been reported in Brazil. Between 1996 and 2009, 42 cases of yellow fever (YF) were registered in the State of Amazonas, and 71.4% (30/42) were fatal. Since 2010, no cases have been reported. Because the introduction of the yellow fever virus into a large city such as Manaus, which is widely infested by Aedes mosquitoes, may pose a real risk of a yellow fever outbreak, efforts to maintain an appropriate immunization policy for the populace are critical. Manaus has also suffered silent outbreaks of Mayaro and Oropouche fevers lately, most of which were misdiagnosed as dengue fever. The tropical conditions of the State of Amazonas favor the existence of other arboviruses capable of producing human disease. Under this real threat, represented by at least 4 arboviruses producing human infections in Manaus and in other neighboring countries, it is important to develop an efficient public health surveillance strategy, including laboratories that are able to make proper diagnoses of arboviruses.

Mitochondrial DNA 4977bp Deletion Mutation in Peripheral Blood Reflects Atrial Remodeling in Patients with Non-Valvular Atrial Fibrillation

PURPOSE: Recently, mitochondrial DNA 4977bp deletion (mtDNA4977-mut), a somatic mutation related to oxidative stress, has been shown to be associated with atrial fibrillation (AF). We hypothesized that patient age, as well as electroanatomical characteristics of fibrillating left atrial (LA), vary depending on the presence of mtDNA4977-mut in peripheral blood among patients with non-valvular AF. MATERIALS AND METHODS: Analyzing clinical and electroanatomical characteristics, we investigated the presence of the mtDNA4977-mut in peripheral blood of 212 patients (51.1+/-13.2 years old, 83.5% male) undergoing catheter ablation for non-valvular AF, as well as 212 age-matched control subjects. RESULTS: The overall frequency of peripheral blood mtDNA4977-mut in patients with AF and controls was not significantly different (24.5% vs. 19.3%, p=0.197). When the AF patient group was stratified according to age, mtDNA4977-mut was more common (47.4% vs. 20.0%, p=0.019) in AF patients older than 65 years than their age-matched controls. Among AF patients, those with mtDNA4977-mut were older (58.1+/-11.9 years old vs. 48.8+/-11.9 years old, p<0.001). AF patients positive for the mtDNA mutation had greater LA dimension (p=0.014), higher mitral inflow peak velocity (E)/diastolic mitral annular velocity (Em) ratio (p<0.001), as well as lower endocardial voltage (p=0.035), and slower conduction velocity (p=0.048) in the posterior LA than those without the mutation. In multivariate analysis, E/Em ratio was found to be significantly associated with the presence of mtDNA4977-mut in peripheral blood. CONCLUSION: mtDNA4977-mut, an age-related somatic mutation detected in the peripheral blood, is associated with advanced age and electro-anatomical remodeling of the atrium in non-valvular AF.

Insertion-deletions burden in copy number polymorphisms of the Tibetan population.

BACKGROUND: Many studies have been conducted to identify either insertions-deletions (inDels) or copy number variations (CNVs) in humans, but few studies have been conducted to identify both of these forms coexisting in the same region. AIMS AND OBJECTIVES: To map the functionally significant sites within human genes that are likely to influence human traits and diseases. MATERIALS AND METHODS: In this report, we describe an inDel map in the 1051 Tibetan CNV regions obtained through CNV genotyping using Affymetrix Genome-wide single nucleotide polymorphism 6.0 chip. InDel polymorphisms in these copy number polymorphism regions were identified with a computational approach using the 2500 deoxyribonucleic acid sequences obtained from the 1000 Genome Project. RESULTS: The study identified a total of 95935 inDels that range from 1 bp to several bps in length which were found scattered across regulatory regions, exons and in introns of genes underlying the CNVs. A study on the distribution of inDels revealed that the majority of inDels were found in coding regions of the genome than the noncoding, while within the genes, inDels in intron regions were more followed by exonic regions and finally the regulatory regions. CONCLUSION: Study of inDels in CNV regions contribute to the enhanced understanding of the role played by the two variations and their collective influence on the genome. Further, a collection of these inDel genetic markers will aid in genetic mapping, further understanding of the phenotypic variability, identification of disease genes and in detecting novel CNVs.

Cystic fibrosis transmembrane conductance regulator mutations at a referral center for cystic fibrosis / Mutações no gene cystic fibrosis transmembrane conductance regulator em um centro de referência para a fibrose cística

OBJECTIVE: To determine the frequency of six mutations (F508del, G542X, G551D, R553X, R1162X, and N1303K) in patients with cystic fibrosis (CF) diagnosed, at a referral center, on the basis of abnormal results in two determinations of sweat sodium and chloride concentrations. METHODS: This was a cross-sectional study involving 70 patients with CF. The mean age of the patients was 12.38 ± 9.00 years, 51.43% were female, and 94.29% were White. Mutation screening was performed with polymerase chain reaction (for F508del), followed by enzymatic digestion (for other mutations). Clinical analysis was performed on the basis of gender, age, ethnicity, pulmonary/gastrointestinal symptoms, and Shwachman-Kulczycki (SK) score. RESULTS: All of the patients showed pulmonary symptoms, and 8 had no gastrointestinal symptoms. On the basis of the SK scores, CF was determined to be mild, moderate, and severe in 22 (42.3%), 17 (32.7%), and 13 (25.0%) of the patients, respectively. There was no association between F508del mutation and disease severity by SK score. Of the 140 alleles analyzed, F508del mutation was identified in 70 (50%). Other mutations (G542X, G551D, R553X, R1162X, and N1303K) were identified in 12 (7.93%) of the alleles studied. In F508del homozygous patients with severe disease, the OR was 0.124 (95% CI: 0.005-0.826). CONCLUSIONS: In 50% of the alleles studied, the molecular diagnosis of CF was confirmed by identifying a single mutation (F508del). If we consider the analysis of the six most common mutations in the Brazilian population (including F508del), the molecular diagnosis was confirmed in 58.57% of the alleles studied. .

OBJETIVO: Determinar a frequência de seis mutações (F508del, G542X, G551D, R553X, R1162X e N1303K) em pacientes com fibrose cística (FC) de um centro de referência, diagnosticados pela presença de duas dosagens de sódio e cloro no suor alteradas. MÉTODOS: Estudo de corte transversal com 70 pacientes com idade média de 12,38 ± 9,00 anos, sendo que 51,43% eram do sexo feminino, e 94,29% eram caucasoides. A triagem de mutações foi realizada pela técnica de reação em cadeia da polimerase (F508del), seguida por digestão enzimática (demais mutações). A análise clínica foi realizada utilizando as variáveis sexo, idade, etnia, manifestações pulmonares/digestivas e escore de Shwachman-Kulczycki (ESK). RESULTADOS: Todos os pacientes apresentaram manifestações pulmonares, e 8 não apresentaram manifestações digestivas. Os resultados do ESK evidenciaram doença leve, moderada e grave, respectivamente, em 22 (42,3%), 17 (32,7%) e 13 (25,0%) pacientes. Não houve associação da mutação F508del com o grau de doença pelo ESK. Dos 140 alelos analisados, a mutação F508del foi identificada em 70 (50%). As demais mutações (G542X, G551D, R553X, R1162X e N1303K) foram identificadas em 12 (7,93%) dos alelos analisados. Em pacientes homozigotos F508del com doença grave, a OR foi de 0,124 (IC95%: 0,005-0,826). CONCLUSÕES: O diagnóstico molecular de FC foi confirmado pela identificação de apenas uma mutação (F508del) em 50% dos alelos estudados. Se considerarmos a análise das seis mutações de maior frequência na população brasileira (incluindo F508del), o diagnóstico molecular foi confirmado em 58,57% dos alelos analisados. .

Type IA isolated growth hormone deficiency (IGHD) consistent with compound heterozygous deletions of 6.7 and 7.6 Kb at the GH1 gene locus / Deficiência isolada de hormônio do crescimento tipo IA (DIGH) consistente com deleções heterozigotas compostas de 6,7 e 7,6 Kb no locus gênico GH1

Isolated growth hormone deficiency (IGHD) may result from deletions/mutations in either GH1 or GHRHR genes. The objective of this study was to characterize the molecular defect in a girl presenting IGHD. The patient was born at 41 weeks of gestation from non-consanguineous parents. Clinical and biochemical evaluation included anthropometric measurements, evaluation of pituitary function, IGF-I and IGFBP-3 levels. Molecular characterization was performed by PCR amplification of GH1 gene and SmaI digestion of two homologous fragments flanking the gene, using genomic DNA from the patient and her parents as templates. At 1.8 years of age the patient presented severe growth retardation (height 61.2 cm, -7.4 SDS), truncal obesity, frontal bossing, doll face, and acromicria. MRI showed pituitary hypoplasia. Laboratory findings confirmed IGHD. GH1 gene could not be amplified in samples from the patient while her parents yielded one fragment of the expected size. SmaI digestion was consistent with the patient being compound heterozygous for 6.7 and 7.6 Kb deletions, while her parents appear to be heterozygous carriers for either the 6.7 or the 7.6 Kb deletions. We have characterized type IA IGHD caused by two different GH1 gene deletions, suggesting that this condition should be considered in severe IGHD, even in non-consanguineous families. Arq Bras Endocrinol Metab. 2012;56(8):558-63.

A deficiência isolada do hormônio do crescimento (DIGH) pode ser resultado de deleções/mutações no gene GH1 ou no gene GHRHR. O objetivo deste estudo foi caracterizar o defeito molecular em uma menina que apresenta DIGH. A paciente nasceu às 41 semanas de gestação de pais não consanguíneos. As avaliações clínica e bioquímica incluíram medidas antropométricas, avaliação da função pituitária e concentrações de IGF-I e IGFBP-3. A caracterização molecular foi feita por meio de amplificação do GH1 por PCR e digestão com SmaI de dois fragmentos homólogos flanqueando o gene, usando-se DNA genômico da paciente e de seus pais como padrões. Com 1,8 ano de idade, a paciente apresentou atraso grave no crescimento (altura 61,2 cm, -7.4 DP), obesidade central, protuberância frontal, face de boneca e acromicria. A RM mostrou hipoplasia pituitária. Os achados laboratoriais confirmaram a DIGH. O gene GH1 não pôde ser amplificado nas amostras da paciente, enquanto as amostras de seus pais produziram um fragmento do tamanho esperado. A digestão com SmaI foi consistente com a paciente ser heterozigota composta para deleções para 6,7 e 7,6 Kb, enquanto seus pais parecem ser carreadores heterozigotos para deleções de 6,7 ou 7,6 Kb. Caracterizamos a DIGH tipo IA causada por duas deleções diferentes no gene GH1, sugerindo que essa condição pode ser considerada na DIGH grave, mesmo em famílias não consanguíneas. Arq Bras Endocrinol Metab. 2012;56(8):558-63.

Utility of molecular studies in incontinentia pigmenti patients.

The diagnosis of incontinentia pigmenti (IP) is fairly easy in the presence of classical features, but can be difficult in cases with partial or non-classical features, especially in the parents. The demonstration that the disease is caused by mutations in the NEMO gene, has remarkably improved genetic counselling for this disorder. We present four families of IP in whom molecular studies established an unequivocal diagnosis in the affected daughters, and showed two mothers to be carriers, thus allowing accurate genetic counselling and prenatal diagnosis.

Insulin-dependent suppression of cholesterol 7alpha-hydroxlase is a possible link between glucose and cholesterol metabolisms

Cholesterol 7alpha-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin-Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.

Insulin-dependent suppression of cholesterol 7alpha-hydroxlase is a possible link between glucose and cholesterol metabolisms

Cholesterol 7alpha-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin-Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.

Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.

Deletional mutations of dystrophin gene and carrier detection in eastern India.

Objective. To determine the pattern of deletions of the dystrophin gene, the major class of mutations among the Duchenne and Becker muscular dystrophy patients of eastern India and to analyze the carrier frequency of the female members of the proband’s family. Methods. Deletional mutations occurring in patients have been characterized by multiplex polymerase chain reaction. Carrier state of mothers and sisters of probands were analyzed by either of two methods: 1) typing polymorphic short tandem repeat markers in or around the regions of deletion, by radioactive polymerase chain reaction and 2) quantitative real time amplification of the region of deletion. Results. Deletions were detected in 67 (62.04%) out of 108 male patients, about 76.12% of these being localized in the central hot spot region of the gene, i.e., between exon 42 to exon 53 and 17.91% at the proximal hot spot i.e., between exon 1 to exon 20. In the present study were found 43 types of deletions, out of which 25 (58%) were new deletions, which were not described earlier among the Indian patients. Distribution pattern of deletions in different hot spot regions has been compared with that of other countries and statistical analysis reveals significant difference between countries (p<0.001). Correlation of the pattern of deletion with clinical phenotype of patients has been discussed. Interesting case of germline mosaicism and its implications in counseling has also been discussed. Conclusion. About half the mothers of affected probands were not carriers of the deletion, underscoring the need to use real time techniques for carrier detection.

Occurrence of Aggregatibacter actinomycetemcomitans in Brazilian indians from Umutina Reservation, Mato Grosso, Brazil

Aggregatibacter actinomycetemcomitans is associated with periodontal disease, especially localized aggressive periodontitis, produces a potent leukotoxin and its distribution is influenced by ethnic characteristics of the population. Objective: Using culture and polymerase chain reaction (PCR) techniques, this study evaluated the occurrence of this microorganism and the distribution of leukotoxic strains isolated from Indians belonging to the Umutima Reservation, Mato Grosso, Brazil. MATERIAL AND METHODS: Forty-eight native Brazilians with gingivitis and 38 with chronic periodontitis, belonging to Umutina, Paresi, Bororo, Bakairi, Kayabi, Irantxe, Nambikwara and Terena ethnicities, were studied. Subgingival, supragingival and saliva samples of each patient were collected and transferred to VMGA III medium and to ultra pure Milli Q water. Bacteria were grown on TSBV agar and incubated in anaerobiosis (90 percent N2 + 10 percent CO2) at 37ºC for 72 h. The presence of the ltx promoter was determined by PCR, and a 530 bp deletion in the promoter was evaluated by using specific primers. RESULTS: A. actinomycetemcomitans was isolated from 8.33 percent of saliva, supragingival and subgingival samples from patients with gingivitis and from 18.42 percent of saliva and supragingival biofilm, and 26.32 percent subgingival biofilm from patients with chronic periodontitis. By PCR, the bacterial DNA was detected in 8.33 percent of saliva, supragingival and subgingival biofilms from patients with gingivitis and from 23.68 percent of saliva, 28.95 percent supragingival biofilm and 34.21 percent subgingival biofilm from patients with periodontitis. All strains were grouped as non-JP2 clones based on the absence of deletion in the leukotoxin promoter. Differences among the microbial and clinical parameters in patients were analyzed by using the Mann-Whitney, Chi-square or Fisher's exact tests. CONCLUSIONS: The present results suggest that A. actinomycetemcomitans ...

Less DeltamtDNA4977 than normal in various types of tumors suggests that cancer cells are essentially free of this mutation

Levels of mtDNA(4977) deletions (DeltamtDNA(4977)) have been found to be lower in tumors than in adjacent non-tumoral tissues. In 87 cancer patients, DeltamtDNA(4977) was detected by multiplex polymerase chain reaction (PCR) amplification in 43 (49%) of the tumors and in 74 (85%) of the samples of non-tumoral tissues that were adjacent to the tumors. DeltamtDNA(4977) deletions were detected in 24% of the breast tumors, 52% of the colorectal tumors, 79% of the gastric tumors, and 40% of the head and neck tumors as compared with 77, 83, 100, and 90% of the adjacent respective non-tumoral tissues at the same DNA template dilution. Based on limiting dilution PCR of 16 tumors and their adjacent non-tumoral tissues, it was found that the amount of DeltamtDNA(4977) was 10- to 100-fold lower in the tumor than in the respective control non-tumoral tissues. Real-time PCR experiments were performed to quantify the number of DeltamtDNA(4977) deletions per cell, by determining the mitochondrial-to-nuclear DNA ratio. In all of the cases of breast, colorectal, gastric, and head and neck cancer the proportion of DeltamtDNA(4977) in tumors was lower than that of the respective non-tumoral tissue. Traces of DeltamtDNA(4977) in tumors were apparently due to contamination of tumor tissue with surrounding non-tumoral tissue, as evidenced by tumor microdissection and in situ PCR techniques, suggesting that tumors are essentially free of this mutation. Although the metabolic effect of DeltamtDNA(4977) may be minimal in normal (non-tumor) tissue, in tissue under stress, such as in tumors, even low levels of DeltamtDNA(4977) deletions may be intolerable.

Weakening of the repressive YY-1 site on the thrombospondin-1 promoter via c-Jun/YY-1 interaction

Thrombospondin-1 (TSP-1) level is tightly regulated at the transcriptional level. To determine the detailed molecular mechanisms of TSP-1 expression, nine serial 5'-deletion constructs of the human genomic tsp-1 promoter (nucleotides -2,220 to +756) were prepared, inserted into luciferase reporter plasmids, and transiently transfected into the Hep3B human hepatocarcinoma cell. Among the nine 5'-deletion constructs, pTSP-Luc-4 (-767~+756) had consistently decreased luciferase activity with or without PMA stimulation, whereas a further truncated construct [pTSP-Luc-4' (-407~+756)] had increased levels of expression. By searching the nucleotides from -767 to -407, a consensus binding sequence (5'-CCATTTT-3') for the repressor Yin Yang-1 (YY-1) at nucleotide -440 was identified. The suppression induced by this site was weakened in the presence of the region upstream of nucleotide -767 (pTSP-Luc-1 and -2). Nuclear protein directly bound to an oligonucleotide containing the repressive YY-1 sequence but the binding capacity of the sequence was decreased by the increased c-Jun levels. Moreover, proteins immunoprecipitated with anti-YY-1 revealed an interaction between c-Jun and YY-1 factor. These data suggest that the repressive YY-1 site of the tsp-1 promoter could not be functional via activating positive cis-elements on the upstream from this site and weakened via c-Jun/YY-1 interactions.

Molecular diagnosis of Prader-Willi syndrome.

BACKGROUND: Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia and feeding problems in infancy, developmental delay, hyperphagia with obesity, short stature, hypogonadism, characteristic facial appearance, and behavior problems. The diagnosis of PWS is based on clinical findings that change with age. PWS has proved to be a difficult condition to recognize with the diagnosis often being delayed until later childhood or even adulthood. Therefore, a molecular testing for PWS is needed to confirm the diagnosis. OBJECTIVE: To study the clinical features of Prader-Willi syndrome patients and confirm diagnosis by molecular testing. MATERIAL AND METHOD: Eighteen Prader-Willi syndrome patients who were diagnosed between March, 1997 and February, 2002 at the Genetic Unit, Queen Sirikit National Institute of Child Health, Bangkok. Peripheral blood lymphocytes were obtained and cultured using the standard technique for chromosome analysis. For fluorescence in situ hybridization (FISH) studies, the specific DNA probes for loci small nuclear ribonucleoprotein polypeptide N (SNRPN) were used to detect deletion. Non-deleted cases were confirmed to have PWS by methylation analysis. RESULTS: The diagnosis of eighteen PWS patients was confirmed by FISH using DNA probes for loci SNRPN demonstrating a deletion of chromosome 15q11-q13 in fourteen cases (77%). Four cases (23%) were confirmed to have PWS resulting from maternal uniparental disomy by demonstrating exclusively maternal specific DNA methylation patterns. CONCLUSION: The clinical diagnosis of PWS should be confirmed by molecular testing especially in the infancy period to avoid needless invasive diagnostic testing.

De novo mutations in sporadic deletional Duchenne muscular dystrophy (DMD) cases

Dinucleotide repeat polymorphism based genetic analysis is a powerful approach to gain insight into rare genetic events like germline mosaicism and de novo mutations. The loss of heterozygosity of polymorphic dinucleotide loci at "deletional hotspot" of dystrophin gene can provide direct evidence of carrier status in female relatives of affected DMD patients with overlapped exonic deletions. We have used 4 STR loci of the central deletional hotspot of the dystrophin gene for genetic analysis in sporadic unrelated DMD families. Twenty-nine mothers of sporadic deletional cases were analysed and their carrier status was determined. Eighteen of them showed heterozygosity in the deleted loci suggesting the occurrence of de novo mutations. In 9 cases, the carrier status was indeterminate while 2 showed germline mosaicism. Our observations reiterated the importance of STR analysis in determining the status of mothers of sporadic deletional DMD cases in order to provide proper genetic counselling.