Rothmund-Thomson Syndrome: in Search of a New Gene

Project location: ITALY
Project start date: December 2007 - Project end date: December 2008
Project number: 2007-14
Beneficiary: Università degli Studi di Milano

The combined strategy of autozygosity mapping, array capture of the candidate interval and Next Generation DNA sequencing enabled to identify the causative mutation in a family affected with Clericuzio-type poikiloderma with neutropenia (PN). The mutation falls within the predicted gene of unknown function C16orf57.
Pedigree: the deleterious allele could have been transmitted to the affected in the fifth generation either via yellow/green pathway, or via blue/red pathway, or via blue/purple pathway.The three siblings of a five-generation inbred Italian family (Figure 1) were initially misdiagnosed with Rothmund-Thomson (RTS [MIM #268400]), a progeria-like syndrome showing phenotypic overlap with Clericuzio-type poikiloderma with neutropenia (PN [MIM %604173]). Both syndromes are rare autosomal-recessive genodermatosis characterised by poikiloderma and short stature, but differential diagnosis is based on radial defects and alopecia (RTS patients), vs pachyonychia and severe chronic neutropenia (PN patients Figure 2).
 PN Phenotype: Affected proband (V-2) and sib (V-4) at age 22 and 14 showing facial poikiloderma extending to buttocks and legs and pachyonychia of the toes.
As the pedigree suggested an autosomal recessive transmission model, we genotyped the affected members as well as all the living subjects of the family. Linkage analysis pointed to a 3.4Mb region on chromosome 16q (Table 1) This was the largest genomic interval inherited identically by descent (IBD) according to an autosomal recessive model. The three affected family members inherited two copies of the same region. Parents were carriers as well as grandparents, uncles and unaffected brothers were either carrier or not carrier, but none of them shared with the affected the same two copies of this region.
Results of genome-wide linkage analysis using 262K NspI SNP Array (Affymetrix) platform, showing three regions with LOD score >2.5This genomic region hosts at least 80 genes, and none was a suitable candidate gene for PN. The standard process of sequencing candidate genes seemed an extremely costly and time consuming procedure. We decided to bypass this step using the new technology of targeted next generation DNA sequencing.
DNA was extracted from one affected patient, and the 3.4Mb target region was enriched in vitro using a chip (Agilent) trapping only the sequences that hybridized against the region of interest. We omitted from the target DNA region all the highly repeated sequences reducing the region to be sequenced to 1.7Mb. Following all the enrichment steps the targeted DNA was re-sequenced using an Illumina platform.
Flow chart of the array capture and deep sequencing procedureThe stepwise procedure is reported (Figure 3). The output of the new sequencing method consists in two files reporting all the mismatches of the input sequence compared against public databases; one file records all the homozygous mismatches and the second the heterozygous mismatches. According to our model, we considered only the homozygous SNP file.
Out of the >1000 homozygous mismatches to the human reference sequence, only 17 were both previously unreported SNPs and mapped close to or within genes. We ranked the substitution according to their evolutionary conservation (Table 2). One of these 17 substitutions alters a splice acceptor sequence for the C16orf57 gene and results in out-of-frame exon skipping in the patient's RNA (Figs. 4, 5).
We validated the role of C16orf57 gene as a second unrelated patient with PN, who was shown to be a compound heterozygote for C16orf57 mutations (Figs. 4, 5).



Figure 4 (above) C16orf57 Mutational Analysis
(A) Schematic genomic structure of C16orf57 gene (16q13) spanning 20Kb and 7 exons (dark boxes) encoding a 265 aa protein. Red arrows point to the identified mutations (primers and conditions are reported in Table S3).
(B and E) Pedigrees and sequencing of C16orf57 genomic mutations: (B) shows the carrier status of both parents and the homozygous splicing mutation c.504-2A>C of the three affected sibs and (E) the parental origin and the two mutations c.666_676+1del12/c.502A>G of the compound heterozygote sporadic patient. Base changes are in red characters.
(C, F, H) Agarose gel electrophoresis showing products of different RT-PCR (primers and conditions are reported in Table S4), on lymphoblastoid cell lines RNA. Lengths of normal and aberrant products are indicated. The following abbreviations are used: V-2 homozygous patient; P compound heterozygous patient; C+ normal control; C- negative control, M Gene Ruler TM DNA Ladder mix (Fermentas).
(D, G, I) Sequence data of the two PN patients showing aberrant transcripts due to out of frame skipping of exon 5, in frame skipping of exon 6 and exon 4, respectively, and related schematic diagrams of the mutation-associated miss-spliced cDNAs.

Figure 5 (above) Graphical view of predicted domain structure of C16orf57protein The green cylinders represent α helices, the yellow arrows β sheets and the thin black lines putative coils. The protein segments encoded by the seven C16orf57 exons are indicated by red dashed lines. (A) Wild type C16orf57 protein (NP_078874)..(B) C16orf57 protein sequence, predicted by mutation c.504-2A>C present in the homozygous condition in the affected sibs of the inbred family. Upon skipping of exon 5 and frameshift, a truncated protein lacking 61 residues (boxed in purple) results (C). (D) Altered proteins predicted by in frame loss of exon 4 and exon 6 encoded domains due to missense c.502A>G and c.666_676+1del12 mutations of the PN compound heterozygous patient. 

Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene. Volpi L, Roversi G, Colombo EA, Leijsten N, Concolino D, Calabria A, Mencarelli MA, Fimiani M, Macciardi F, Pfundt R, Schoenmakers EF, Larizza L.
Am J Hum. Genet. 2010 Jan;86(1):72-6. Epub 2009 Dec 10.

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