Evidence that SIZN1 is a candidate X-linked mental retardation gene

Abstract

An estimated 1-3% of individuals within the United States are diagnosed with mental retardation (MR), yet the cause is unknown in nearly 50% of the patients. While several environmental, genetic and combined teratogenetic etiologies have been identified, many causative genes remain to be identified. Furthermore, the pathogenetic mechanisms underlying MR are known for very few of these genes. Males have a much higher incidence of MR implicating genes on the X-chromosome. We have recently identified a novel gene, SIZN1, on the X-chromosome and showed that it functions in modulating the BMP signaling pathway. Furthermore, we have shown this gene is necessary for basal forebrain cholinergic neuron (BFCN) specific gene expression. Given that cognitive function is impaired when BFCNs are lost or functionally disrupted, we undertook a screen of cognitively impaired males for SIZN1 mutations. We report on four different sequence variants in SIZN1 in 11 individuals with nonsyndromic X-linked mental retardation (XLMR). Our data implicate SIZN1 as a candidate gene for XLMR and/or as a neurocognitive functional modifier.

Figure 1

A. Schematic diagram of the human X chromosome showing the SIZN1(ZCCHC12) locus. B. Genome structure on human X-chromosome. SIZN1 has 4 exons with the entire coding region contained within exon 4. C. Alignment between mouse and human Sizn1 protein. Location of sequence variations, a putative NLS (nuclear localization sequence) and a CCHC-type zinc finger motif at the C-terminus are indicated.

Figure 2

X chromosome marker analysis and X inactivation (Xi) data are shown for the c.19C>T(p.R7C) sequence variation in SIZN1. (A) c.19C>T (p.R7C) was found in family, K9264. Mutation analysis in family K9264 and two unrelated patients with MR (CMS4957 and CMS7492). A 622-bp SIZN1 exon 4 PCR product amplified from genomic DNA was digested with Nsp1 restriction enzyme to distinguish between the normal (622-bp black arrowhead) and the mutant (555-bp red arrowhead) alleles. A 67-bp fragment generated by the restriction digestion in affected individuals is not shown. Restriction digestion analysis shows affected males carry the c.19C→T mutation. (B) The p.R7C mutant Sizn1 shows reduced activity in SBEx4 reporter assay. Western blot analysis of the SIZN1 protein shows the reduced activity correlates with reduced protein levels (approximately 40% reduction; n=8). (C) Pulse-chase analysis of Sizn1 Proteins (n=3). Error bar are standard deviation (SD) in luciferase assay.

Figure 3

SIZN1 mutation c.1031C>T(p.T344I) in family K8923 (A). Partial pedigree of family K8923 with XLMR chromosome marker analysis and X inactivation (Xi) data are indicated. (B) Automated sequence chromatograms of SIZN1 exon 4 from a control male and from two affected males and a carrier female in family K8923 showing a “C” to “T” alteration at nucleotide 1031 (c.1031C→T). Carrier mother is heterozygous for the alteration. Mutant alleles are boxed. This alteration is predicted to cause a p.T344I missense change in SIZN1. (C) Sequence comparison across species shows the Thr 344 residue is highly conserved amino acid. (D) The Sizn1 related enhancement in BMP signaling is suppressed by the T344I mutant of Sizn1 (30%) in SBEx4 reporter assay. Error bar are standard deviation (SD) in luciferase assay.