1 Analysis Of ZRS Mutations In Two Families With Triphalangeal Thumbs And Preaxial Polydactyly 2.The Preliminary Genetic Study Of Familial Nonmedullary Thyroid Cancer

Background:Polydactyly is a series of limb malformation with relatively high incidence rate. Preaxial polydactyly (PPD), with the extra finger(s) occuring at radial side, has remarkably high incidence among polydactyly, and may occur as isolated phenotype or part of a syndrome, such as Geig syndrome and Acropectoral syndrome. Triphalangeal thumb (TPT) sometimes accompanies with PPD. PPD Ⅱ-Ⅲ both have the characteristic of TPT, and here named TPT-PPD.Zone of polarizing activity regulatory sequence (ZRS) locates in the fifth intron of gene LMBR1, which is a 1.3kb regulatory sequence that enhances the expression of SHH 1Mb downstream. The expression of SHH in the posterior limb bud marks the zone of polarising activity (ZPA), it regulates the formation of normal anterior-posterior axis pattern. Ectopic anterior expression of SHH at early limb bud stages attributes to the digit malformations, including but not limited to TPT-PS, PPDII, tibial hypoplasia or aplasia with polydactyly (THYP), Laurin Sandrow syndrome (LSS).Objective:To identify the causative mutations in two Chinese Han TPT-PPD families.Methods:Blood samples were collected from 9 members (2 affected) from family 1 and 14 members (7 affected) from family 2. After genomic DNA was extracted, the ZPA regulatory sequence (ZRS) was analyzed with real-time quantitative PCR (qPCR) and Sanger sequencing. ZRS of 200 genomic DNA samples from unrelated individuals are sequenced as controls. For family 1, haplotypes encompassing the ZRS were also analyzed with short tandem repeats (STRs) and single nucleotide variants (SNVs).Results:No copy number variants around ZRS were found in both families as assayed by qPCR. Two heterozygous mutations in the ZRS were found to co-segregate with the TPT-PPD malformation in family 1 (ZRS 406A>G) and 2 (ZRS 105C>G), respectively. Neither mutation was detected in 200 healthy individuals. Haplotype analysis and Sanger sequencing of ZRS in family 1 indicated that the first TPT-PPD patient in the family (Ⅱ4) was both germLine and somatic mosaic for the ZRS 406A>G mutation.Conclusion:Two pathogenic ZRS mutations, ZRS 406A>G and ZRS 105C>G, have been identified in two Chinese Han families with TPT-PPD, among which the ZRS 406A>G mutation was de novo.Background:Thyroid cancer (TC) is the most common malignant pathological changes in the endocrine system. The morbidity of TC grows rapidly in china and over the world. TC is classified into two major types through cell origin:follicle origin medullary thyroid cancer (MTC), and C-cell origin non-medullary thyroid cancer (NMTC). NMTC accounts for 95% of TC, and one of the subtypes of NMTC, papillary thyroid cancer (PTC), accounts for 85% of NMTC.4.2% of NMTC is familial, with 2 or more patients in one family, thus called familial non-medullary thyroid cancer (FNMTC), The prognosis of FNMTC is obviously worse than sporadic NMTC, and requires more aggressive treatment. As for the genetic study, there are merely several candidate genes that had been identified by linkage analysis within families or by genome-wide association study (GWAS). The pathogenic genetic variations of the disease remain unclear.Objective:To identify the inherited pathogenic germline mutations of FNMTC through the study of 10 FNMTC families (family 1-10).Methods:1) On the premise that all members involved in this study understood and signed the informed consent, we obtained the venous blood of the members and extracted the genome DNA. 2) For 3 healthy and 1 affected individuals in family 2, SNP array is performed to identify the copy number variations (CNVs). Call the CNVs that cosegregate with NMTC phenotype as candidates responsible for the phenotype.3) For the same individuals in method 2, whole exome sequencing (WES) is performed to identify the co-segregating mutations within this family. All the variants identified by WES went throught the pipeline constructed on the basis of population frequency, conservation, and protein function. The remaining variants were validated by PCR-Sanger sequencing and then a list of the candidate causative genes was generated.4) One patient from each of the other 9 families is selected to preform PCR-Sanger sequencing to see if there is any mutation in those candidate genes. All the family members went through PCR-Sanger sequencing and co-segregation analysis if mutation in the candidate genes was identified in the selected patient from the same family.5) HABP2 was reported as a candidate gene of FNMTC, so PCR-Sanger sequencing was performed in all the patients from 10 FNMTC families to identify HABP2 mutations in its 3’UTR,5’UTR, exons and the flanking intronic-regions.Results:1) No suspicious CNV in family 2 was found by the SNP array test.2) 13 candidate genes were obtained from the WES data in family2:ERBB3, GTF2F2, GALC, MPP4. CCDC150, SPRYD3, C14orfl69, GSE1, MNAT1, TRIP 12, FMNL3, DNAH7, and NCL.3) Two mutations of the candidate genes were found in the probands of other two families:TRIP 12 C.561C>T heterozygous mutation in family 7, and GSE c.3491-3495del heterozygous mutation in family 8. Further sequencing results of the other members in family 7 and 8 didn’t show the mutation-phenotype co-segregation in each of their families.4) No suspicious mutation in HABP2 s was found in 10 FNMTC families.