Catherine J. Bromhead

Kufs Disease, the Major Adult Form of Neuronal Ceroid Lipofuscinosis, Caused by Mutations in CLN6

The molecular basis of Kufs disease is unknown, whereas a series of genes accounting for most of the childhood-onset forms of neuronal ceroid lipofuscinosis (NCL) have been identified. Diagnosis of Kufs disease is difficult because the characteristic lipopigment is largely confined to neurons and can require a brain biopsy or autopsy for final diagnosis. We mapped four families with Kufs disease for whom there was good evidence of autosomal-recessive inheritance and found two peaks on chromosome 15. Three of the families were affected by Kufs type A disease and presented with progressive myoclonus epilepsy, and one was affected by type B (presenting with dementia and motor system dysfunction). Sequencing of a candidate gene in one peak shared by all four families identified no mutations, but sequencing of CLN6, found in the second peak and shared by only the three families affected by Kufs type A disease, revealed pathogenic mutations in all three families. We subsequently sequenced CLN6 in eight other families, three of which were affected by recessive Kufs type A disease. Mutations in both CLN6 alleles were found in the three type A cases and in one family affected by unclassified Kufs disease. Mutations in CLN6 are the major cause of recessive Kufs type A disease. The phenotypic differences between variant late-infantile NCL, previously found to be caused by CLN6, and Kufs type A disease are striking; there is a much later age at onset and lack of visual involvement in the latter. Sequencing of CLN6 will provide a simple diagnostic strategy in this disorder, in which definitive identification usually requires invasive biopsy.

Human and Mouse Mutations in WDR35 Cause Short-Rib Polydactyly Syndromes Due to Abnormal Ciliogenesis

Defects in cilia formation and function result in a range of human skeletal and visceral abnormalities. Mutations in several genes have been identified to cause a proportion of these disorders, some of which display genetic (locus) heterogeneity. Mouse models are valuable for dissecting the function of these genes, as well as for more detailed analysis of the underlying developmental defects. The short-rib polydactyly (SRP) group of disorders are among the most severe human phenotypes caused by cilia dysfunction. We mapped the disease locus from two siblings affected by a severe form of SRP to 2p24, where we identified an in-frame homozygous deletion of exon 5 in WDR35. We subsequently found compound heterozygous missense and nonsense mutations in WDR35 in an independent second case with a similar, severe SRP phenotype. In a mouse mutation screen for developmental phenotypes, we identified a mutation in Wdr35 as the cause of midgestation lethality, with abnormalities characteristic of defects in the Hedgehog signaling pathway. We show that endogenous WDR35 localizes to cilia and centrosomes throughout the developing embryo and that human and mouse fibroblasts lacking the protein fail to produce cilia. Through structural modeling, we show that WDR35 has strong homology to the COPI coatamers involved in vesicular trafficking and that human SRP mutations affect key structural elements in WDR35. Our report expands, and sheds new light on, the pathogenesis of the SRP spectrum of ciliopathies.

Reducing the exome search space for Mendelian diseases using genetic linkage analysis of exome genotypes

Many exome sequencing studies of Mendelian disorders fail to optimally exploit family information. Classical genetic linkage analysis is an effective method for eliminating a large fraction of the candidate causal variants discovered, even in small families that lack a unique linkage peak. We demonstrate that accurate genetic linkage mapping can be performed using SNP genotypes extracted from exome data, removing the need for separate array-based genotyping. We provide software to facilitate such analyses.

GFI1B mutation causes a bleeding disorder with abnormal platelet function

GFI1B is a transcription factor important for erythropoiesis and megakaryocyte development but previously unknown to be associated with human disease.

Generating linkage mapping files from Affymetrix SNP chip data

SUMMARY LINKDATAGEN is a perl tool that generates linkage mapping input files for five different linkage mapping tools using data from all 11 HAPMAP Phase III populations. It provides rudimentary error checks and is easily amended for personal linkage mapping preferences. AVAILABILITY AND IMPLEMENTATION LINKDATAGEN is available from http://bioinf.wehi.edu.au/software/linkdatagen/ with accompanying annotation files, reference manual and test dataset.

Variable hearing impairment in a DFNB2 family with a novel MYO7A missense mutation

Hildebrand MS, Thorne NP, Bromhead CJ, Kahrizi K, Webster JA, Fattahi Z, Bataejad M, Kimberling WJ, Stephan D, Najmabadi H, Bahlo M, Smith RJH. Variable hearing impairment in a DFNB2 family with a novel MYO7A missense mutation.

Mutations in TMC1 are a common cause of DFNB7/11 hearing loss in the Iranian population.

Objectives: We investigated the cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) that segregated in 2 consanguineous Iranian families. Methods: Otologic and audiometric examinations were performed on affected members of each family. Genome-wide parametric multipoint linkage mapping using a recessive model was performed with Affymetrix 50K GeneChips or short tandem repeat polymorphisms. Direct sequencing was used to confirm the causative mutation in each family. Results: In 2 Iranian families, L-1651 and L-8600606, with ARNSHL that mapped to the DFNB7/11 locus, homozygosity for a reported splice site mutation (c.776+1G>A), and a novel deletion (c.1589_1590delCT; p.S530) were identified in the TMC1 gene, respectively. Conclusions: Consistent with the previously reported phenotype in DFNB7/11 families, the 2 Iranian families had segregated congenital, profound hearing impairment. However, in family L-1651, one affected family member (IV:3) has milder hearing impairment than expected, suggesting a potential genetic modifier effect. These results indicate that DFNB7/11 is a common form of genetic hearing loss in Iran, because this population is the source of 6 of the 29 TMC1 mutations reported worldwide.

Mutations in SH3PXD2B cause Borrone dermato-cardio-skeletal syndrome

Borrone Dermato-Cardio-Skeletal (BDCS) syndrome is a severe progressive autosomal recessive disorder characterized by coarse facies, thick skin, acne conglobata, dysmorphic facies, vertebral abnormalities and mitral valve prolapse. We identified a consanguineous kindred with a child clinically diagnosed with BDCS. Linkage analysis of this family (BDCS1) identified five regions homozygous by descent with a maximum LOD score of 1.75. Linkage analysis of the family that originally defined BDCS (BDCS3) identified an overlapping linkage peak at chromosome 5q35.1. Sequence analysis identified two different homozygous mutations in BDCS1 and BDCS3, affecting the gene encoding the protein SH3 and PX domains 2B (SH3PXD2B), which localizes to 5q35.1. Western blot analysis of patient fibroblasts derived from affected individuals in both families demonstrated complete loss of SH3PXD2B. Homozygosity mapping and sequence analysis in a second published BDCS family (BDCS2) excluded SH3PXD2B. SH3PXD2B is required for the formation of functional podosomes, and loss-of-function mutations in SH3PXD2B have recently been shown to underlie 7 of 13 families with Frank-Ter Haar syndrome (FTHS). FTHS and BDCS share some overlapping clinical features; therefore, our results demonstrate that a proportion of BDCS and FTHS cases are allelic. Mutations in other gene(s) functioning in podosome formation and regulation are likely to underlie the SH3PXD2B-mutation-negative BDSC/FTHS patients.

A Novel Splice Site Mutation in the RDX Gene Causes DFNB24 Hearing Loss in an Iranian Family

To the Editor: Hearing impairment is the most common genetic sensory defect in humans worldwide. It is estimated that profound hearing loss occurs in 4 out of every 10,000 children [Morton 1991]. Seventy percent of hereditary SNHL is nonsyndromic (DFN) and in at least 80% of these cases the inheritance is autosomal recessive (DFNB) [Smith et al., 2005]. Genetic studies have shown that mutations in 67 loci and 25 genes are associated with DFNB [Van Camp and Smith, 2008]. Further understanding of the molecular mechanisms associated with SNHL will assist in the development of therapeutics for hereditary hearing loss in the future. Mutations in the RDX gene that encodes the radixin protein have recently been shown to cause DFNB24 hearing loss in consanguineous Pakistani and Indian families [Khan et al., 2007]. In this study, we report a novel splice site mutation in the RDX gene in a consanguineous Iranian family with autosomal recessive non-syndromic hearing loss (ARNSHL). This mutation is only the fourth to be identified at the rare DFNB24 ARNSHL locus and the second that is predicted to lead to nonsense mediated decay or a truncated version of the radixin protein. A five-generation Iranian family with apparent congenital ARNSHL was studied (Fig.1). Family members in the fourth and fifth generations had severe-to-profound hearing loss suggesting autozygosity by descent (Fig.2). A complete physical examination and audiologic testing was completed on consenting family members and blood samples were obtained for DNA extraction. Liver function testing on affected individual V:2 showed total bilirubin (0.4 mg/dl), direct bilirubin (0.1 mg/dl), alanine transaminase (41 U/ml), aspartate transaminase (35 U/ml) and alkaline phosphatase (229 U/L), all normal. Human Research Institutional Review boards at the Welfare Science and Rehabilitation University and Iran University of Medical Sciences and the University of Iowa approved all procedures. Fig. 1 Pedigree of the Iranian family with ARNSHL. Open symbols = unaffected; filled symbols = ARNSHL. The genotypes and haplotypes for affected individuals and carriers are shown. Fig. 2 Audiograms from affected family members. All affected individuals displayed severe-to-profound bilateral ARNSHL. Genomic DNA from individuals III:1, IV:3, IV:4, IV:5, IV:6, IV:7, IV:8, V:1, V:2, V:3, and V:6 (Fig.1) was genotyped for 50,000 SNPs using Affymetrix (Santa Clara, CA, USA) 50K XBA GeneChips at the Translational Genomics Research Institute (Pheonix, Arizona). All genotypes were determined using the BRLMM genotyping algorithm [Di et al., 2005; Rabbee and Speed 2006]. Genotyping data were examined with PEDSTATS [Wigginton and Abecasis 2005] for Mendelian inheritance errors and MERLIN [Abecasis et al., 2002] for errors based on inferred double recombination events between tightly linked markers. Because the size of the pedigree was above the threshold for efficient implementation of the Lander-Green multipoint linkage mapping algorithm, a smaller subset of the pedigree was used which only considered the descendents of individuals III:1, III:2, III:3, III:4 (Fig.1). An autosomal, genome-wide parametric linkage analysis was carried out as the pedigree showed affected males and females. All linkage analyses were performed with MERLIN. The Lander-Green multipoint linkage mapping algorithm assumes linkage equilibrium. The markers were too close together to be in linkage equilibrium, so a smaller marker set was created using an in-house program (linkdatagen.pl;http://bioinf.wehi.edu.au/ software/). The binsize was set to 0.5 cM (i.e. the highest heterozygosity marker was chosen in each 0.5 cM interval) and 6140 autosomal markers were selected. Linkage analyses were carried out using the following parameters for the disease allele a, and normal allele A - Pr(a)=0.0001, Pr(disease|aa)=0.999, Pr(disease|AA or Aa)=0.001 - reflecting a highly penetrant, autosomal recessive disease model. The genetic map used was generated by Affymetrix using interpolation based on the empirical deCode, Marshfield and SNP Linkage (SLM1) genetic maps [Broman et al., 1998; Kong et al., 2002]. The physical map used was derived from the UCSC database using the NCBI Build 36.1 (March 2006; http://genome.ucsc.edu/cgi-bin/hgGateway). In the genome-wide analysis 38 Mendelian (.07% error rate) and 168 double recombinant errors were detected. A peak LOD score of 4.13 was achieved for a region on chromosome 11 (Fig.3) and there were no other regions where the LOD score exceeded 3. The critical region spanned approximately 3.6 cM between markers rs1789819 (109,185,632 bp) and rs10502160 (112,122,938 bp) (Fig.1). All four affected individuals included in the mapping were homozygous for a region between 108.814 cM and 112.368 cM on chromosome 11 (11q22.3-23.1). Fig. 3 Graphic showing the only significant LOD score achieved in the genome-wide analysis on chromosome 11. The dashed line indicates threshold (LOD score of 3). The dotted line at LOD score of 3.6 represents the Lander-Kruglyak threshold. The peak LOD score ... The critical region contained the known ARNSHL locus DFNB24. The RDX gene was amplified and sequenced using previously reported gene-specific primers [Khan et al., 2007]. A novel homozygous splice site mutation (c.698+1G>A) in intron 7 was found to segregate in affected family members (Fig.4). The +1G nucleotide of the 5’ donor splice site is invariant among all eukaryotes [Shapiro and Senapathy, 1987]. The c.698+1G>A mutation is predicted to result in read-through into intron 7 and a stop codon immediately following the amino acids encoded by exon 7. The last residue encoded by exon 7 would remain a lysine (K) due to the redundancy of the genetic code (AAG > AAA) and the next codon created would be a stop (TAA). This is predicted to result in a severely truncated protein lacking part of the FERM3 domain and the entire α- and c-terminal (CTD) domains (Fig.5), although the mutant mRNA may be subject to nonsense mediated decay and in this case no mutant protein would be produced. The c.698+1G>A mutation was not observed in 53 (106 chromosomes) Iranian and 133 (266 chromosomes) CEPH control individuals. Fig. 4 Sequence chromatograms showing the novel c.698+1G>A mutation in the RDX gene. Fig. 5 Domain structure of the RDX gene showing mutations known to cause DFNB24 hearing loss in humans. The novel splice site mutation described in this study (c.698+1G>A) is boxed. Radixin and ezrin are present in the hair cell stereocilia of the mouse inner ear [Kitajiri et al., 2004; Pataky et al., 2004]. While it is known that radixin functions as a linker between sub-cellular actin and the plasma membrane [Niggli and Rossy 2008], the exact role of this protein in the stereocilia of hair cells is unclear. Current knowledge of the role of these proteins in the inner ear comes from study of a radixin knockout mouse model. RDX −/− mice exhibit syndromic hearing impairment and hyperbilirubinemia [Kikuchi et al., 2002; Kitajiri et al., 2004]. The murine liver phenotype does not appear to model the human phenotype. One affected individual in the family reported here and another in a previously reported DFNB24 family have been shown to have normal liver function [Khan et al., 2007]. In conclusion, we have identified the first splice site mutation in the RDX gene and the first mutation known to cause DFNB24 hearing loss in the Iranian population. Our data also suggest that, in contrast to the murine model, liver dysfunction is not a component of the human phenotype. DFNB24 appears to be a rare form of ARSNHL as only four families have been reported. Further characterization of the molecular events triggered by this new mutation may increase our understanding of the function of radixin protein in the inner ear.

Complete callosal agenesis, pontocerebellar hypoplasia, and axonal neuropathy due to AMPD2 loss

Objective: To determine the molecular basis of a severe neurologic disorder in a large consanguineous family with complete agenesis of the corpus callosum (ACC), pontocerebellar hypoplasia (PCH), and peripheral axonal neuropathy. Methods: Assessment included clinical evaluation, neuroimaging, and nerve conduction studies (NCSs). Linkage analysis used genotypes from 7 family members, and the exome of 3 affected siblings was sequenced. Molecular analyses used Sanger sequencing to perform segregation studies and cohort analysis and Western blot of patient-derived cells. Results: Affected family members presented with postnatal microcephaly and profound developmental delay, with early death in 3. Neuroimaging, including a fetal MRI at 30 weeks, showed complete ACC and PCH. Clinical evaluation showed areflexia, and NCSs revealed a severe axonal neuropathy in the 2 individuals available for electrophysiologic study. A novel homozygous stopgain mutation in adenosine monophosphate deaminase 2 (AMPD2) was identified within the linkage region on chromosome 1. Molecular analyses confirmed that the mutation segregated with disease and resulted in the loss of AMPD2. Subsequent screening of a cohort of 42 unrelated individuals with related imaging phenotypes did not reveal additional AMPD2 mutations. Conclusions: We describe a family with a novel stopgain mutation in AMPD2. We expand the phenotype recently described as PCH type 9 to include progressive postnatal microcephaly, complete ACC, and peripheral axonal neuropathy. Screening of additional individuals with related imaging phenotypes failed to identify mutations in AMPD2, suggesting that AMPD2 mutations are not a common cause of combined callosal and pontocerebellar defects.