Genetic Diagnosis Of Three Cases Of Neurofibromatosis Type Ⅰ Presenting With Multiple Café-au-lait Macules

BackgroundCafe-au-lait macules(CALMs)are pigmented macula or patches with two to three tones of darker skin color than those not involved.The incidence rate is about 10%in the population.About 3%of the newborns have one or more Cafe-au-lait macules.The most typical clinical feature of neurofibromatosis type I are cafe-au-lait macules(usually≥ 6 pieces),and 99%of patients have Cafe-au-lait macules as the first symptom,which appear more than 2 years old.Neurofibromatosis type I(NF1,OMIM:162200)is an autosomal dominant disease and the main clinical features of NF1 were Cafe-au-lait macules,neurofibromas,axillary and inguinal freckles,Lisch nodules and bone damage.In addition to NF1,it may be an early manifestation of genetic syndromes such as Legius syndrome,LEOPARD syndrome,Watson syndrome,and familial progressive hyperpigmentation and hypopigmentation when patients only present with multiple Cafe-au-lait macules.Gene detection can be performed by whole exome sequencing technology to identify diseases.With the wide application of whole exome sequencing technology in gene research,it has been proved to be of great significance in the diagnosis of genetic diseases.ObjectiveWhole exome sequencing was performed on 3 patients with multiple Cafe-au-lait macules,and the results were verified by Sanger sequencing to make a clear diagnosis.Methods1.Case collection:the cases included 2 families and 1 sporadic patient.The clinical data and peripheral blood samples of 3 patients and 4 normal subjects were collected.2.Whole exome sequencing was performed on the peripheral blood DNA of the samples.Firstly,we used PCR-free technology to construct the whole genome library,used the whole exon capture Kit(NanoWES Human Exome,Berry and Kang company),and used IIumina NovaSeq 6000 high-throughput sequencer for high-depth sequencing.3.According to the whole exome sequencing results,Sanger sequencing was performed on the screened loci.Results1.Whole exome sequencing:the whole exome sequencing results of case 1,case 2 and case 3 genomic DNA showed that no pathogenic mutations were found in the pathogenic genes NF2,PTPN11,KRAS,HRAS,NRAS,BRAF,RAF1,SOS1,LZTR1,MAP2K1,MAP2K2,MAS and SPRED1 associated with multiple coffee spots.In case 1,exon 37 of NF1 gene had a missense mutation,and the base at position 5546 was replaced by A from G(c.5546G>A),resulting in the change of amino acid at position 1849 of protein from arginine to glutamine(p.R1849Q).The parents of case 1 did not have the above mutation.In case 2,the missense mutation occurred in exon 13 of NF1 gene,and the base at 1466 was replaced by G from A(c.1466aA>G),resulting in the change of amino acid at position 489 of the protein from tryptophan to cysteine(p.Y489C).The parents of case 2 did not have the above mutation.In case 3,a nonsense mutation occurred in exon 11 of NF1 gene,and the 1246th base was replaced by T from C(c.1246C>T),resulting in the early termination of transcription at position 416 of amino acid(p.R416*).2.Sanger sequencing verification:Sanger sequencing was performed on the loci screened by the whole exome of case 1 and its parents,case 2 and its parents,and case 3 respectively.The Sanger sequencing verification results were consistent with the results of the whole exome.ConclusionIn this study,we performed whole-exome sequencing on 3 patients with clinical manifestations of Cafe-au-lait macules and verified it with Sanger sequencing,all of which were found to have mutations in the NF1 gene and were finally diagnosed as Neurofibromatosis type I.Studies have proved that whole-exome sequencing combined with Sanger sequencing is an effective method for early diagnosis of neurofibromatosis type Ⅰ.