Disruption of the serine/threonine kinase 9 gene causes severe X-linked infantile spasms and mental retardation

Abstract

X-linked West syndrome, also called "X-linked infantile spasms" (ISSX), is characterized by early-onset generalized seizures, hypsarrhythmia, and mental retardation. Recently, we have shown that the majority of the X-linked families with infantile spasms carry mutations in the aristaless-related homeobox gene (ARX), which maps to the Xp21.3-p22.1 interval, and that the clinical picture in these patients can vary from mild mental retardation to severe ISSX with additional neurological abnormalities. Here, we report a study of two severely affected female patients with apparently de novo balanced X;autosome translocations, both disrupting the serine-threonine kinase 9 (STK9) gene, which maps distal to ARX in the Xp22.3 region. We show that STK9 is subject to X-inactivation in normal female somatic cells and is functionally absent in the two patients, because of preferential inactivation of the normal X. Disruption of the same gene in two unrelated patients who have identical phenotypes (consisting of early-onset severe infantile spasms, profound global developmental arrest, hypsarrhythmia, and severe mental retardation) strongly suggests that lack of functional STK9 protein causes severe ISSX and that STK9 is a second X-chromosomal locus for this disorder.

Figure 1

Patient 1 at the age of 3 years.

Figure 2

FISH mapping of the Xp22.3 translocation breakpoints. A, Ideograms of chromosomes X and 7 and their derivatives der(X) and der(7) in patient 1 with 46,X,t(X;7)(p22.3;p15), with indications of breakpoints (arrows). B, Ideograms of chromosomes X and 6 and their derivatives der(X) and der(6) in patient 2 with 46,X,t(X;6)(p22.3;q14). C, FISH analysis of the patient’s chromosomes with the breakpoint-spanning clone RP1-245G19, which shows signals on the normal X chromosome, the der(X), and the der(7) (arrows). Cohybridization with an Xptel-specific cosmid probe was used to indicate the normal and derivative X chromosomes (arrowheads). Arrows point to the breakpoints. D, The metaphase shows the breakpoint-spanning PAC J659D24 with signals on the normal X chromosome and split signals on the very large derivative X and the derivative chromosome 6.

Figure 3

A, Diagram of the genomic structure of the STK9 gene and selected clones used for breakpoint mapping by FISH. Exons (unshaded boxes) and introns are not drawn to scale. The respective positions of the two breakpoints studied (t(X;7)(p22.3;p15) and t(X;6)(p22.3;q14)) are indicated on top. The lower panel shows a restriction map of the area surrounding both breakpoints with probes used for Southern blot hybridization indicated as solid rectangles. Position and size of the normal restriction fragments detected by the two probes is indicated (B = BamHI, E = EcoRI, H = HindIII, EV = EcoRV). B, Southern blot analysis, using the probes indicated as solid rectangles in the lower panel of A. Patient [(tX;6) and t(X;7)] and control DNAs (F = female, M = male) were digested with the indicated enzymes. Rearranged (junction) fragments (arrows) are only present in the patients but not in the controls. C, RT-PCRs in patients’ lymphoblastoid (tX;6) and fibroblast (tX;7) RNAs and control RNAs (C = control lymphoblastoid cell line RNA in case of t(X;6), C = control fibroblast RNA in case of t(X;7), iBr mRNA = infant brain mRNA, gDNA = genomic DNA, M = size marker). Left side of this panel shows a specific STK9 amplification product of 208 bp (primers STK9.1 and STK9.2, see “Subjects and Methods” section) in all control RNAs but not in patient RNA. The low molecular weight band visible in almost all lanes represents the primer dimer product. The esterase D (ESD) gene served as a control for patient cDNA established from mRNA and total RNA. Right side shows specific RT-PCR products of 678 bp with a primer set spanning STK9 exons 1–10 in both patient and control RNA. Primers spanning the breakpoint (located in exons 9 and 12) amplify STK9 transcripts of 1029 bp in the control but not in the patient. D, Chromosome X, 7, der(X) and der(7) sequences at the breakpoints in patient 1. Chromosome X-derived sequences are shown in bold. The breakpoints are on either side of the common 3 bp-sequence (shaded boxes).