Combined cytogenetic and molecular analyses for the diagnosis of Prader-Willi/Angelman syndromes.

Prader-Willi (PWS) and Angelman (AS) are syndromes of developmental impairment that result from the loss of expression of imprinted genes in the paternal (PWS) or maternal (AS) 15q11-q13 chromosome. Diagnosis on a clinical basis is difficult in newborns and young infants; thus, a suitable molecular test capable of revealing chromosomal abnormalities is required. We used a variety of cytogenetic and molecular approaches, such as, chromosome G banding, fluorescent in situ hybridization, a DNA methylation test, and a set of chromosome 15 DNA polymorphisms to characterize a cohort of 27 PWS patients and 24 suspected AS patients. Molecular analysis enabled the reliable diagnosis of 14 PWS and 7 AS patients, and their classification into four groups: (A) 6 of these 14 PWS subjects (44 %) had deletions of paternal 15q11-q13; (B) 4 of the 7 AS patients had deletions of maternal 15q11-q13; (C) one PWS patient (8 %) had a maternal uniparental disomy (UPD) of chromosome 15; (D) the remaining reliably diagnoses of 7 PWS and 3 AS cases showed abnormal methylation patterns of 15q11-q13 chromosome, but none of the alterations shown by the above groups, although they may have harbored deletions undetected by the markers used. This study highlights the importance of using a combination of cytogenetic and molecular tests for a reliable diagnosis of PWS or AS, and for the identification of genetic alterations.

Direct deletion analysis in two Duchenne muscular dystrophy symptomatic females using polymorphic dinucleotide (CA)n loci within the dystrophin gene.

Duchenne muscular dystrophy (DMD) is the most common hereditary neuromuscular disease. It is inherited as an X-linked recessive trait in which males show clinical manifestations. In some rare cases, the disease can also be manifested in females. The aim of the present study was to determine the molecular alteration in two cases of nonrelated DMD symptomatic carriers with no previous history of DMD. Multiplex PCR is commonly used to search for deletion in the DMD gene of affected males. This method could not be used in females because the normal X chromosome masks the deletion of the mutated one. Therefore, we used a set of seven highly polymorphic dinucleotide (CA)(n) repeat markers that lie within the human dystrophin gene. The deletions were evidenced by hemizygosity of the loci under study. We localized a deletion in the locus 7A (intron 7) on the maternal X chromosome in one case, and a deletion in the region of introns 49 and 50 on the paternal X chromosome in the other. The use of microsatellite genotyping within the DMD gene enables the detection of the mutant allele in female carriers. It is also a useful method to provide DMD families with more accurate genetic counseling.