Ataxia-Pancytopenia Syndrome Is Caused by Missense Mutations in SAMD9L

Abstract

Ataxia-pancytopenia (AP) syndrome is characterized by cerebellar ataxia, variable hematologic cytopenias, and predisposition to marrow failure and myeloid leukemia, sometimes associated with monosomy 7. Here, in the four-generation family UW-AP, linkage analysis revealed four regions that provided the maximal LOD scores possible, one of which was in a commonly microdeleted chromosome 7q region. Exome sequencing identified a missense mutation (c.2640C>A, p.His880Gln) in the sterile alpha motif domain containing 9-like gene (SAMD9L) that completely cosegregated with disease. By targeted sequencing of SAMD9L, we subsequently identified a different missense mutation (c.3587G>C, p.Cys1196Ser) in affected members of the first described family with AP syndrome, Li-AP. Neither variant is reported in the public databases, both affect highly conserved amino acid residues, and both are predicted to be damaging. With time in culture, lymphoblastic cell lines (LCLs) from two affected individuals in family UW-AP exhibited copy-neutral loss of heterozygosity for large portions of the long arm of chromosome 7, resulting in retention of only the wild-type SAMD9L allele. Newly established LCLs from both individuals demonstrated the same phenomenon. In addition, targeted capture and sequencing of SAMD9L in uncultured blood DNA from both individuals showed bias toward the wild-type allele. These observations indicate in vivo hematopoietic mosaicism. The hematopoietic cytopenias that characterize AP syndrome and the selective advantage for clones that have lost the mutant allele support the postulated role of SAMD9L in the regulation of cell proliferation. Furthermore, we show that AP syndrome is distinct from the dyskeratoses congenita telomeropathies, with which it shares some clinical characteristics.

Figure 1

Identification of SAMD9L Mutations in Two Families Affected by Autosomal-Dominant AP Syndrome (A) Family UW-AP, with the c.2640C>A mutation. The segregation of microsatellite-defined haplotypes on 7q21–7q31 is shown for all pedigree members from whom a blood sample was obtained. The location of SAMD9L is indicated on the STRP schematic, shown per the Marshfield map. Recombination events in individuals III-6 and IV-2 define a 32 Mb region on the red haplotype; missing genotypes from connecting relatives preclude use of individual III-1 to further narrow the region. Exome sequencing was performed on individuals IV-1 and IV-3. (B) Family Li-AP, with the c.3587G>C mutation. Filled-in quadrants indicate the variable manifestations in both pedigrees; d. denotes age at death; c. denotes current age.

Figure 2

Imaging and Pathology of the AP Syndrome (A) Cerebellar section of individual UW-AP III-5 after death from a retroperitoneal bleed at age 16 years, stained with anti vimentin (scale bar, 100 μm). This demonstrates marked loss of Purkinje cells and milder loss of granule cells. (B) T-1-weighted sagittal MRI of the brain of individual UW-AP III-8 at age 38, showing moderate midline cerebellar atrophy despite mild clinical manifestations. (C) T-1-weighted sagittal MRI of individual Li-AP II-4 at age 52, showing marked midline cerebellar and pontine atrophy. (D) T-2-weighted coronal MRI of individual Li-AP II-4 at age 52, showing diffuse bilateral abnormal white matter signal.

Figure 3

Genotyping and Linkage Analysis in the UW-AP Family (A) SNPs on chromosome 7 demonstrating LOH for most of the long arm in a cell line from III-3. Minor-allele, b, frequency (MAF) in III-3 (red) is compared to MAF in an affected relative whose DNA was analyzed at the same time (black). (B) Log R ratio of SNP signals show equivalent signal intensities across chromosome 7 for individual III-3 and his relative, consistent with diploidy. (C) SNPs on chromosome 7 demonstrating LOH for most of the long arm in an LCL from UW-AP individual IV-3. MAF in the LCL (red) is compared to MAF in his buffy coat DNA (black), analyzed at the same time. (D) Log R ratio of SNP signals in individual IV-3 shows equivalent signal intensities across chromosome 7 for both of his samples, consistent with diploidy. (E) SNP genome LOD plot of UW-AP. The LCL sample from individual III-3 was omitted from the analysis with Allegro without loss of power because genotypes from his parents and affected children were included. There were five regions of interest with LOD scores greater than 1.5.

Figure 4

Allele-Specific PCR Amplification of the c.2640C>A Mutation in Blood and LCLs in the UW-AP Family The mutant allele was amplified with primers containing the mutant nucleotide, A, in the 3′ position and a mismatched nucleotide, T, in the preceding position. In unaffected individual III-2 no mutant band was visualized. In blood from all three affected individuals, IV-1, III-3 and IV-3, a mutant band is seen. In culture of LCLs from III-3 and IV-3, the mutant band is diminished, as shown for time points T1 to T3.

Figure 5

Response of UW-AP LCLs to DNA Damaging Agents Individual UW-AP III-2 is unaffected, UW-AP IV-3 is affected, NC is a normal control, and FA is a patient with Fanconi anemia. (A) The surviving fraction of treated cells compared to untreated cells after 5 days of exposure to MMC (n = 3, mean ± SEM). (B) The surviving fraction of treated cells compared to untreated cells 5 days after a dose of IR (n = 3, mean ± SEM). (C) Quantification by Western blot of proteins involved in induction of the FA pathway or in ATM or ATR signaling. Cells were harvested 8 hr after treatment with IR (10 Gy) or 24 hr after continuous exposure to MMC (60 ng/mL), as indicated. In contrast to the increased sensitivity to DNA damaging agents manifested by FA, response of IV-3 was not different from that of III-2 or NC.