Diagnosis Of Patients With Cornelia De Lange Syndrome By Cytogenetic And Molecular Genetic Techniques

Background Cornelia de Lange syndrome (CdLS) (OMIM #122470, #300590 and #610759) is a rare dominant genetic disorder with multiple organ system abnormalities which is classically characterized by typical facialfeatures, growth and mental retardation, upper limb defects(ranging from small hands and subtle phalangeal and metacarpal changes to severe forms of oligodactyly and forearm truncation primarily involving the ulnar structures), hirsutism, gastrointestinal and other visceral system involvement [1]. The facial feature includes synophrys, long eyelashes, a depressed nasal root with an up-turned nasal tip and anteverted nares, a long philtrum, a thin upper lip, small widely spaced teeth, brachymicro-cephaly, low-set and posteriorly rotated ears [1]. Other common findings include ptosis, myopia, intestinal malrotation, cryptorchidism, hypospadias, pyloric stenosis, congenital diaphragmatic hernias, cardiac septal defects, seizures and hearing loss. Mental retardation is typically severe; IQs range from less than 30 to 102 with an average of 53 [1]. Heterozygous mutations in the cohesin regulator, NIPBL (50-60%), or the cohesin structural components SMC1A and SMC3 (5%), have been identified in approximately 65% of individuals with CdLS [1] [2].Objective Four patients with Cornelia de Lange Syndrome from four Chinese families were recruited in the State Key Laboratory of Medical Genetics of China. We use cytogenetic and a variety of molecular genetic methods to study 4 probands, to provide diagnosis and genetic counseling for the probands and their families, and to provid gene diagnosis guidance for the other CdLS patients.Methods Chromosomal analysis was done on the peripheral blood lymphocytes of the probands according to conventional techniques and high resolution banding analysis. Mutational analysis of the NIPBL, SMC1A and SMC3 gene are carried out by polymerase chain reaction (PCR), reverse transcription PCR, direct sequencing in the probands, apply SNP array to detect the genome-wide copy number variations. DNAs from parents were sequenced in the corresponding region when the mutation was detected in their infected child. The new mutation was detected in 100 normal individuals by direct sequencing.Results The chromosomal analysis revealed all the probands with a normal karyotypes.We found no mutations in proband 1, SNP Array analysis found no pathogenic copy number variations; there was a heterozygous mutation c.4321G>T in the exon 20 of the NIPBL gene in proband 2, which was previously reported; we detected a heterozygous mutation c.6589+5G>C in the intron 38 of the NIPBL gene in proband 3, which was absent in her parents. We carried out a RT-PCR to understand the splicing of the exon 38, found that this mutation caused a splicing mutation in the exon 38, generated both normal transcript and an aberrant alternatively spliced transcript with an exon 38 deletion. The C.6589+5G >C mutation is absent in 100 normal individuals. No mutations were detected in proband 4.Conclusions The NIPBL gene heterozygous mutation c.4321G>T and c.6589+5G>C are the disease-causing mutation of the proband 2 and the proband 3 respectively, the c.4321G>T was previously reported, and the c.6589+5G>C is a novel splicing mutation that has not been reported. We confirmed two patients with Cornelia de Lange Syndrome in China by mutational analysis.