CASK mutations are frequent in males and cause X-linked nystagmus and variable XLMR phenotypes

Abstract

Mutations of the calcium/calmodulin-dependent serine protein kinase (CASK) gene have recently been associated with X-linked mental retardation (XLMR) with microcephaly, optic atrophy and brainstem and cerebellar hypoplasia, as well as with an X-linked syndrome having some FG-like features. Our group has recently identified four male probands from 358 probable XLMR families with missense mutations (p.Y268H, p.P396S, p.D710G and p.W919R) in the CASK gene. Congenital nystagmus, a rare and striking feature, was present in two of these families. We screened a further 45 probands with either nystagmus or microcephaly and mental retardation (MR), and identified two further mutations, a missense mutation (p.Y728C) and a splice mutation (c.2521-2A>T) in two small families with nystagmus and MR. Detailed clinical examinations of all six families, including an ophthalmological review in four families, were undertaken to further characterise the phenotype. We report on the clinical features of 24 individuals, mostly male, from six families with CASK mutations. The phenotype was variable, ranging from non-syndromic mild MR to severe MR associated with microcephaly and dysmorphic facial features. Carrier females were variably affected. Congenital nystagmus was found in members of four of the families. Our findings reinforce the CASK gene as a relatively frequent cause of XLMR in females and males. We further define the phenotypic spectrum and demonstrate that affected males with missense mutations or in-frame deletions in CASK are frequently associated with congenital nystagmus and XLMR, a striking feature not previously reported.

Figure 1

Reverse transcriptase PCR (RT-PCR) analysis on Family 74. (a) RT-PCR analysis of cDNA from a lymphoblastoid cell line from the proband of family 74 and wild-type control. Forward primer was designed from exon 21, 5′-CTGGCATTCGATTCAGAGTTGGTG-3′, and reverse primer from exon 24, 5′-CGCAAACCGGTCTGGGTGCTTTG-3′. Lanes 1 and 3: affected male and control male, respectively; Lanes 2 and 4: no RT controls. (b) Sequence traces from the lymphoblastoid cell line showing the 27 bp deletion compared with control lymphoblastoid cell line cDNA. The dotted line indicates the deleted bases in the abnormal transcript.

Figure 2

Schematic representation of the full-length CASK gene showing the different domains and both previously reported (in red) and new mutations (in black). The mutation nomenclature is based on the CASK full-length isoform encoding 926 amino acids (ENST00000378163). The four mutations in parentheses at the C-terminal end are associated with nystagmus.

Figure 3

Family pedigrees of the six families with mutations in the CASK gene. Males are indicated by boxes and females by circles. Individuals with intellectual disability are shown as black boxes and circles. Carriers (either obligate or by testing) are shown with a dot within the circle. The letter ‘N' in parentheses indicates individuals with nystagmus. The horizontal bar above the symbols indicates individuals who underwent DNA analysis. Mutant (^*) and wild-type (wt) alleles are indicated below the symbols.

Figure 4

Protein alignment of CASK orthologues. Homo sapiens (ENSP00000367405), Pan troglotydes (ENSPTRP00000047442), Macaca mulatta (ENSMMUP00000041117), Mus musculus (ENSMUSP00000112322), Rattus norvegicus (ENSRNOP00000004187), Canis familiaris (ENSCAFP00000021243), Bos taurus (ENSBTAP00000026825), Gallus gallus (ENGALP000000026115), Xenopus tropicalis (ENSXET00000010527), Danio rerio (ENSDARP00000091622), Drosophila melanogaster (FBPP0083559) and Caenorhabditis elegans (F17E.5.1a). Missense mutations are indicated by arrows and red letters. Amino acids that are not conserved are shown in blue. The protein 4.1 binding motif (Hook motif) is underlined in the Homo sapiens sequence. The deleted amino acids (p.710_718) in family 74 are indicated by bracket.

Figure 5

Reverse transcriptase PCR analysis of Family 683. (a) Lane 1: affected male; Lane 2: obligate carrier; Lane 3: wild-type control; and Lane 4: negative control. Forward primer was designed from exon 24, 5′-TCATCACAAAGCACCCAGAC-3′, and reverse primer was from the 3′-UTR, 5′-CCATGACCTGCTGACACAAG-3′. (b) Sequence traces showing the wild-type junction sequence between exons 25–26 and exons 26–27 and the two abnormal transcripts observed in family 683 as a result of splice mutation (c.2521-2A>T). The dotted line indicates the deleted bases in the abnormal transcript.

Figure 6

Faces of affected individuals with dysmorphic facial features. (a–c) Family V: The proband, II-4 (a), and his brother, II-2 (b), both show microcephaly, high nasal bridge, upslanting palpebral fissures and a short columella. Their sister, II-3 (c), is non-dysmorphic.