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ORIGINAL ARTICLE. Carol Dobson-Stone . Antonio Velayos-Baeza . An Jansen . Frederick Andermann . François Dubeau . Francine Robert . Anne Summers .
Neurogenetics (2005) 6: 151–158 DOI 10.1007/s10048-005-0220-9

ORIGINA L ARTI CLE

Carol Dobson-Stone . Antonio Velayos-Baeza . An Jansen . Frederick Andermann . François Dubeau . Francine Robert . Anne Summers . Anthony E. Lang . Sylvain Chouinard . Adrian Danek . Eva Andermann . Anthony P. Monaco

Identification of a VPS13A founder mutation in French Canadian families with chorea-acanthocytosis Received: 5 April 2005 / Accepted: 27 April 2005 / Published online: 26 May 2005 # Springer-Verlag 2005

Abstract Mutations in VPS13A cause chorea-acanthocytosis (ChAc), an autosomal recessive neurodegenerative disorder. VPS13A is located in a tail-to-tail arrangement with GNA14 on chromosome 9q21. ChAc shows substantial allelic heterogeneity, with no single VPS13A mutation causing the majority of cases. We examined 11 patients in four French Canadian ChAc pedigrees for mutations in VPS13A. Affected members of three families were homozygous for a 37-kb deletion of the four terminal exons of VPS13A (EX70_EX73del). This deletion also encompasses the two terminal exons of GNA14. Two affected females in family 4 were homozygous for the splicing mutation 4242+ 1G>T. Remarkably, the affected males in this highly consanguineous pedigree were compound heterozygotes for EX70_EX73del and 4242+1G>T. PCR analysis of the deletion breakpoint junction revealed that an additional patient with French Canadian ancestry was heterozygous for the EX70_EX73del allele. The identification of a common 9q21 haplotype associated with EX70_EX73del

in at least four apparently unrelated ChAc families implies that ChAc shows a founder effect in French Canadians, and that routine testing for EX70_ EX73del in suspected ChAc cases may therefore be worthwhile in this population. The deletion breakpoint PCR described here will enable rapid identification of both homozygous and heterozygous carriers of EX70_EX73del. Keywords Neuroacanthocytosis . Founder effect . DNA mutational analysis . Nucleic acid repetitive sequences . G alpha subunit 14 Abbreviations ChAc: chorea-acanthocytosis . DHPLC: denaturing high-performance liquid chromatography . FRAM: free right-arm monomer . MER: medium reiteration frequency element . OPMD: oculopharyngeal muscular dystrophy . SNP: single nucleotide polymorphism

C. Dobson-Stone . A. Velayos-Baeza . A. P. Monaco (*) The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK e-mail: [email protected] Tel.: +44-1865-287502 Fax: +44-1865-287650

A. E. Lang Division of Neurology, University of Toronto, Toronto, Canada

A. Jansen . F. Andermann . F. Dubeau Department of Neurology and Neurosurgery, McGill University, and the Montreal Neurological Hospital and Institute, Montreal, Canada

A. Danek Neurologische Klinik, Ludwig-Maximilians-Universität, Munich, Germany

S. Chouinard André-Barbeau Movement Disorders Unit, University of Montreal, Montreal, Canada

F. Robert Genetics Program, North Bay & District Health Unit, North Bay, Canada

E. Andermann Department of Neurology and Neurosurgery, and Department of Human Genetics, McGill University, and the Montreal Neurological Hospital and Institute, Montreal, Canada

A. Summers Medical Genetics, North York General Hospital, Department of Paediatrics, University of Toronto, Toronto, Canada

C. Dobson-Stone The Garvan Institute of Medical Research, University of New South Wales, Sydney, Australia

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Introduction

Patients and methods

Chorea-acanthocytosis (ChAc, OMIM 200150) is a neurodegenerative disorder characterised by gradual onset of hyperkinetic movements and aberrant erythrocyte morphology. Onset of neurological symptoms is usually in the third to fifth decade, and the disease follows a progressive course [1, 2]. A few kindreds with apparent autosomal dominant inheritance of ChAc have been reported (e.g. [3, 4]) but most cases are consistent with autosomal recessive transmission. In addition to choreatic limb movements in patients with ChAc, the orofacial region is usually affected and can cause tongue and lip biting, dysarthria and dysphagia. Other movement abnormalities include motor and vocal tics, limb or axial dystonia and parkinsonism [5]. About half of patients with ChAc have seizures [5] and personality and behavioural changes and cognitive deficits are common [6]. We and others identified CHAC (now renamed VPS13A [7]) as the gene mutated in ChAc [8, 9]. VPS13A is organised in 73 exons spanning about 240 kb on chromosome 9q21. VPS13A encodes chorein, a 360-kDa protein that is absent or markedly reduced in patients with ChAc [10]. To date, 75 different VPS13A mutations have been reported in 58 ChAc probands, indicating substantial allelic heterogeneity in this disorder [8–11]. The French Canadian population is of great interest to geneticists. It was founded by approximately 8,500 settlers in the 17th century and has since increased in relative isolation to more than six million people in the province of Quebec [12]. Many mendelian diseases occur at unusually high frequency in its subpopulations, for example, oculopharyngeal muscular dystrophy (OPMD, OMIM 164300) is found worldwide but is at greatest concentration in Northeastern Quebec. Analysis of haplotypes in 42 French Canadian OPMD families pointed to a single OPMD-associated haplotype in the patients, indicating a founder effect for this disorder [13]. Similarly, a common ancestral haplotype was identified in French Canadian juvenile haemochromatosis patients [14]; and a single >15kb deletion in the LDLR gene was found to cause hypercholesterolemia in more than 60% of French Canadian cases [15]. So far, only one French Canadian family has been reported with molecularly diagnosed ChAc. The affected family members harboured a homozygous deletion of the last four exons of VPS13A (EX70_EX73del) [11]. In this study, we report the VPS13A mutation analysis of three additional French Canadian ChAc pedigrees, the further characterisation of the EX70_EX73del mutation and investigation of the founder origin of this deletion in five ChAc pedigrees with French Canadian ancestry.

Subjects Four French Canadian families segregating ChAc were studied. Patient 1-1 corresponds to proband 42 in a previous study [11]. A clinical report has been published for patients 1-1 and 1-2 [16]. Cognitive and ocular motor findings have been reported on patient 4-2 (case 1 in [17]; case 8 in [6]). The diagnosis of ChAc was based on the presence of acanthocytes and/or on characteristic clinical findings in patients with a movement disorder in whom McLeod syndrome had been excluded. All families originate from different regions of the province of Quebec. Eleven patients in six sibships had clinical features of ChAc. Four of the six sibships had clear parental consanguinity (Fig. 1). The main clinical features of the patients are summarised in Table 1. Epilepsy was the presenting feature in five patients, all of whom had confirmed temporal lobe epilepsy. Two additional patients also showed seizures, and age at onset of seizures ranged from 20 to 38 years. Seizures preceded the onset of other clinical manifestations of ChAc by up to 15 years. The other presenting features were movement disorder (four patients), dysarthria and psychiatric symptoms (one patient each). Abnormal movements such as tics, tongue protrusion, chorea, dystonia or parkinsonism were present in all patients except in one who was diagnosed relatively recently (patient 3-3). Carbamazepine and lamotrigine worsened abnormal movements, whereas phenytoin and phenobarbital were better tolerated. Drooling and dysphagia, present in eight patients, resulted in eating difficulties and represented a severe social handicap. Neuropsychological testing commonly revealed full-scale IQ in the low average range, impaired verbal and nonverbal memory and frontal lobe dysfunction. Psychiatric problems included emotional flattening or lability, disinhibition, anxiety, depressive mood or psychosis. Brain imaging showed caudate atrophy in five of eight patients tested. Peripheral blood was collected for DNA isolation after patients had given informed consent in accordance with local ethics guidelines. Mutation analysis Extraction of genomic DNA from peripheral blood leukocytes, amplification of VPS13A exons and mutation analysis using denaturing high-performance liquid chromatography (DHPLC) was performed as described previously [11]. For identification of the 4242+1G>T mutation, VPS13A exon 36 and flanking intronic sequence were amplified using the following primers: 5′-GCCAGA AGTTACTGAGTTTTAC-3′ & 5′-TTCTCAGAGGGAC

153

a

b

Family 1

5

Family 4

4

VPS13A ex

69 70-73 GNA14 ex 7-6 5

1

2

3

+ +

+ +

+ + + +

+ + + +

+ + + +

11 Family 3

Family 2

1

* * VPS13A ex

69 70-73 GNA14 ex 7-6 5 chorein

1 + + + + +

*

2

3

4

+ + [-]

+ +

+ +

*

1 + + + + +

*

2

*

+ + [-]

G/G

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G/T [-]

G/T

G/T

5

6

7

G/T

G/G

T/T

*

8

9

10

T/T -

G/G

G/G

G/T

+ + [-]

Fig. 1 a, b Identification of VPS13A mutations in French Canadian ChAc families. Family members who were not analysed are outlined in grey. Chorein detection was performed on erythrocyte membrane preparations from asterisked individuals. Presence (+), marked reduction ([−]), or absence (−) of chorein is indicated below each individual analysed. a Simplified pedigree structures of families 1–

3. Presence (+) or absence (−) of VPS13A exons 69–73 and GNA14 exons 7–5 is indicated below each individual analysed. In family 2, the parents of 2-2 and 2-3 are second-degree cousins; the parents of 2-4 are third-degree cousins. b Simplified pedigree structure of family 4. The genotype at the 4242+1 site is indicated below each family member analysed

AGATACTAC-3′. Amplicons were sequenced in both directions by means of the BigDye Terminator version 3.1 Cycle Sequencing Ready Reaction kit, and samples were subjected to capillary electrophoresis on the ABI PRISM 3770 (both from Applied Biosystems, Warrington, UK). GNA14 exons 1–7 were amplified as described previously [18].

For analysis of the EX70_EX73del deletion breakpoint junction, the following primer pairs were used: 5′-GTTTG TTGTTGAGACAAGGTCT-3′ and 5′-TTACTGAGGACT CCAAGAGCACTG-3′ (F1–R1, for deletion breakpoint junction) and 5′-TCCATTTGCTGACATTGATTCTC-3′ & 5′-TCAGTGTTCTCATCCCATTCAG-3′ (F2–R2, for positive control). PCRs were carried out in standard 2.5 mM

Table 1 Clinical findings of patients with ChAc from families 1 to 4 Patienta

First symptoms (years) Movement disorder [Age of onset (years)] Tics Chorea Dysarthria Dysphagia Seizures Psychiatric problems Neuropathy (Method) Caudate atrophy a

1-1

1-2

2-2

2-3

2-4

3-2

3-3

4-2

4-3

4-7

4-8

D (33) + (39) + + + NA + + + (Exam) NA

P (17) + (31) + + + + − + + (Exam) NA

S (26) + (39) + + + + + + + (EMG) +

S (23) + (38) + + + + + + − (EMG) +

S (26) + (32) + − + + + + + (EMG) +

S (30) + (36) + + + + + + + (Exam) −

S (38) −

M (15) + (15) + + + + + − + (Exam) +

M (27) + (27) + + + + − − + (Exam) NA

M (38) + (38) + + + NA − + + (EMG) +

M (41) + (41) + + + + − − + (EMG) −

− − + − + − + (Exam) −

Patients are numbered according to the pedigrees shown in Fig. 1 D Dysarthria, P pyschiatric symptoms, S seizures, M movement disorder, + present, − absent, NA information not available, Exam physical examination, EMG electromyography

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MgCl2 reaction mixes containing 0.04 U/μl AmpliTaq Gold (Applied Biosystems). Thermocycling was performed as follows: 95°C for 15 min; 35 cycles of 95°C for 30 s, 62°C (deletion breakpoint junction) or 58°C (control) for 30 s, 72°C for 1 min 30 s; 72°C for 10 min. Deletion junction amplicons were cloned using the pGEMT system (Promega Corporation, Madison, WI, USA) before sequencing as above. Chorein detection Western blot analysis of erythrocyte membrane preparations with anti-chor1 antiserum was performed as previously described [10]. Haplotype analysis Polymorphic microsatellite markers GATA89A11, D9S1674, GATA89C08, D9S153, D9S1780 and D9S1867 flanking VPS13A were used to define haplotypes in the CHAC critical region. Amplification conditions were as described previously [19]. Samples were subjected to capillary electrophoresis on the ABI PRISM 3770 and analysed using the Genotyper program (Applied Biosystems). The following single nucleotide polymorphisms (SNPs) within VPS13A were genotyped to establish haplotypes: 615+20G>C (in intron 8), 3508−51A>G (intron 32), 3508−44G>T (intron 32), 6027C>T (exon 46), 6492T>C (exon 48), 8571T>C (exon 63), 8667+106A>G (intron 63) and 9078−133A>G (intron 68). Exons 8, 33, 46, 48, 63 and 68 and flanking intronic sequence were amplified and directly sequenced as above. Southern blot analysis Southern blot analysis was performed on BamHI, EcoRI and XbaI digests of genomic DNA according to standard procedures [20]. Unique probes were amplified from genomic DNA using the following primer pairs: 5′-TTCCT GTGTCCTTCATTGTGTC-3′ and 5′-TCATGTTCTCATC CCATTCAG-3′ (for probe 1, probe length 500 bp) and 5′TGACGAGGGTCTATTTCCTGAC-3′ and 5′-GGAATG CAGAAAGGATTTACAG-3′ (for probe 2, probe length 685 bp).

70–73 in patients 1-1 and 1-2 [11]. The 7-exon gene encoding Gα subunit 14 (GNA14, OMIM *604397) lies in a tail-to-tail arrangement with VPS13A. PCR analysis of families 1, 2 and 3 revealed that GNA14 exons 6 and 7 could not be amplified in affected family members, implying that the deletion actually extended across the intergenic region between VPS13A and GNA14 and encompassed the last two exons of GNA14 (Fig. 1a). Patient 4-7 is homozygous for the splice-site mutation 4242+1G>T in intron 36, which had previously been identified in an Algerian patient (proband 40 in [11]) and her affected brother [21]. All available family 4 members were screened for the 4242+1G>T mutation by sequence analysis (Fig. 1b). Intriguingly, we discovered that although patient 4-7 and her affected sister (patient 4-8) were homozygous for the splice-site mutation, two affected brothers in a different branch of the family (patients 4-2 and 4-3) carried only one copy of the mutation. Reverse transcriptase PCR analysis revealed that 128 bp, corresponding to exon 36, was deleted in the mutant allele. This leads to a shift in the reading frame and introduction of a stop codon 21 bp downstream of the splice site (data not shown). Absence or marked reduction of chorein was confirmed in several affected family members by Western blot analysis (Fig. 1). Haplotype analysis To establish whether the EX70_EX73del mutation had been inherited from a common ancestor, we performed haplotype analysis of the chromosome 9q21 region in these families. Apart from heterozygosity in patient 2-2 for GATA89c08 (presumably due to a de novo mutation), we discovered that all affected members of families 1, 2 and 3 were homozygous for haplotype A, spanning ∼4 cM (∼2.8 Mb) around VPS13A (Table 2). Genotyping of eight SNPs within VPS13A confirmed these results. As expected, patients 4-7 and 4-8 were homozygous for a different haplotype extending from GATA89a11 to GATA89c08 (haplotype B in Table 2). However, patients 4-2 and 4-3 were heterozygous for haplotypes A and B. This implied that these individuals, who displayed ‘classic’ symptoms of ChAc despite having only one copy of the ‘family’ mutation 4242+1G>T, might be heterozygous in addition for the EX70_EX73del mutation. Further characterisation of EX70_EX73del mutation

Results Mutation screening of VPS13A The entire coding and flanking intronic sequence in VPS13A was screened for mutations by DHPLC in patients 2-2, 3-2 and 4-7. In patients 2-2 and 3-2, it was not possible to amplify exons 70–73, implying that they harboured a homozygous deletion spanning this region. We had previously identified a homozygous deletion of VPS13A exons

Initial attempts to amplify across the deletion breakpoint junction by PCR were unsuccessful, so we investigated the deletion using Southern blot analysis (Fig. 2). Two probes were used: probe 1, located 4.6 kb downstream of VPS13A exon 69, and probe 2, spanning GNA14 exon 5. Although these two regions are 38.6 kb apart in the wild-type allele, they were present on the same BamHI and XbaI restriction fragments in patient 1-1, implying that the deleted region extended for approximately 37 kb. Patient 4-2 showed a hybridisation pattern corresponding to both patient 1-1 and

155 Table 2 Haplotype analysis of the chromosomal region flanking VPS13A in patients with ChAc a

Microsatellite and SNP markers are ordered according to their chromosomal position, with most proximal at the top. D9S1674 is located in VPS13A intron 33. Haplotype A is set in italics; haplotype B is set in bold. A possible mutated allele in patient 2-2 is underlined b Nucleotides are numbered according to the cDNA sequence of VPS13A isoform A as reported by Rampoldi et al. [8] (GenBank accession no. NM_033305), with the adenosine of the initiation codon assigned position 1

Patient 1-1 GATA89a11a 615+20G>Cb 3508−51A>G 3508−44G>T D9S1674 6027C>T 6492T>C 8571T>C 8667+106A>G 9078−133A>G GATA89c08 D9S153 D9S1780 D9S1867

2 G G T 4 C C C G G 2 2 5 4

1-2 2 G G T 4 C C C G G 2 2 5 4

2 G G T 4 C C C G G 2 2 5 4

2-2 2 G G T 4 C C C G G 2 2 5 4

2 G G T 4 C C C G G 1 2 5 4

2-3 2 G G T 4 C C C G G 2 2 5 4

2 G G T 4 C C C G G 2 2 5 4

2-4 2 G G T 4 C C C G G 2 2 5 4

2 G G T 4 C C C G G 2 2 5 4

3-2 2 G G T 4 C C C G G 2 2 5 4

2 G G T 4 C C C G G 2 2 5 4

3-3 2 G G T 4 C C C G G 2 2 5 4

2 G G T 4 C C C G G 2 2 5 4

4-2 2 G G T 4 C C C G G 2 2 5 4

1 C A T 2 T T T A A 2 2 3 3

4-3 2 G G T 4 C C C G G 2 2 5 4

1 C A T 2 T T T A A 2 2 3 1

4-7 2 G G T 4 C C C G G 2 2 5 4

1 C A T 2 T T T A A 2 1 2 4

4-8 1 C A T 2 T T T A A 2 2 3 1

1 C A T 2 T T T A A 2 1 2 4

1 C A T 2 T T T A A 2 2 3 1

wild-type patterns, confirming that this individual was indeed heterozygous for the EX70_EX73del mutation. To further characterise the mutation and to detect additional heterozygous carriers of the deleted allele, we developed a deletion breakpoint junction PCR. The primers span a 38.5-kb region in the wild-type allele. However, in the deleted allele, the primers are located close enough together to generate a 1.2-kb PCR product (Fig. 3). This

confirmed that patients 4-2 and 4-3 were heterozygous for the EX70_EX73del allele. Unaffected individuals 4-1 and 1-4 were also identified as heterozygous carriers of the deleted allele. Sequence analysis revealed that the same 37,278-bp segment is deleted in families 1–4. The deletion junction is located 5,548 bp downstream of VPS13A exon 69 and 896 bp downstream of GNA14 exon 5 (Fig. 3b). The precise description of this mutation is therefore EX69

Fig. 2 a, b Southern blot analysis of VPS13A EX70_73 mutation. a Scaled map of the wild-type locus. The top trace shows the relative positions of VPS13A exons 68–73 and GNA14 exons 7–3; the regions amplified for probes 1 and 2 are shown in grey. The bottom three traces show BamHI, EcoRI and XbaI restriction site positions; expected restriction fragments are labelled a, c and e (for probe 1) and b, d and f (for probe 2). b Hybridisation of probes 1 and 2 to BamHI, EcoRI and XbaI digests of patient 1-1, control (C) and patient 4-2 genomic DNA. Wild-type restriction fragments are

labelled a–f. Restriction fragments originating from the mutant allele are labelled x, y and z and are approximately 7.6, 4.0 and 6.8 kb long, respectively. Due to deletion of the intervening sequence in the mutant allele, probes 1 and 2 hybridise to the same BamHI and XbaI restriction fragments (x and z, respectively). The control individual is heterozygous for duplication of a 71-bp sequence in GNA14 intron 5; consequently, a double band (asterisked) is observed for restriction fragment f

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Fig. 3 a, b Identification of the EX70_EX73del deletion breakpoint junction. a PCR amplification of the region spanning the deletion breakpoint junction. Primer pair F1–R1 produces an amplicon of ∼1,178 bp in individuals who harbour one or two copies of the deleted allele (actual product length varies from 1,176 to 1,179 bp, due to variability in the length of a poly-T tract within the sequence). Primer pair F2–R2 was used as a positive control (amplicon length 1,312 bp). M Molecular weight marker, N DNA-negative control. b Schematic of the genomic region deleted. The region deleted is shown in grey; 5′ and 3′ deletion breakpoints are arrowed. The distribution of Alu (red), FRAM (blue), L2 long interspersed nu-

clear elements (white), mammalian-wide interspersed repeats (yellow), Tigger DNA transposons (brown) and other medium reiteration frequency sequences (green) are shown within two 4-kb regions spanning the deletion breakpoints. Repetitive elements were identified using the RepeatMasker program (http://woody.emblheidelberg.de/repeatmask/). Triangles are aligned according to the orientation of the repeat. Regions amplified by primer pairs F1–R1 and F2–R2 are indicated below the relevant traces. The sequence chromatogram of the deletion breakpoint junction is shown at the bottom of the figure. wt Wild type

+5548_oGNA14:EX5+896del. The 5′ deletion breakpoint occurs within a free right-arm monomer (FRAM) element and the 3′ deletion breakpoint is located within a medium reiteration frequency 5B (MER5B) sequence, 56 bp upstream of an AluY element. The FRAM and AluY elements are in reverse orientation with respect to VPS13A transcription. Previous mutation screening attempts had failed to identify a VPS13A mutation on one or both alleles in 23 patients with suspected ChAc [8, 10, 11] (and unpublished data, 2004). Deletion breakpoint junction PCR analysis of the EX70_EX73del allele in these individuals revealed an additional patient harbouring the deletion (data not shown). This patient (proband 20 in [11]) is also heterozygous for a 1-bp insertion in exon 53, 7339_7340insT, which leads to a shift in the reading frame and introduction of a premature termination codon. In the absence of additional family members, it was not possible to unambiguously determine

phase when genotyping the 9q21 region in this patient. However, the microsatellite and SNP genotypes were consistent with heterozygous inheritance of haplotype A from GATA89a11 to GATA89c08 (data not shown). Inquiries into the patient’s family history revealed that although she was born in the USA, her father is French Canadian (C. Singer 2004, personal communication).

Discussion This study describes the identification of the same deletion of VPS13A exons 70–73 in five apparently unrelated French Canadian ChAc families. VPS13A intron 69 and GNA14 intron 5 are composed of 41 and 34% repetitive sequences, respectively, and it is possible that the deletion was mediated by these elements. The 5′ and 3′ deletion breakpoints occur within a FRAM element and near an

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AluY element, respectively. FRAM elements represent one of the monomeric progenitors of the Alu repeat dimer [22]. Deletions mediated by Alu/Alu recombination events have contributed to many diseases, including hypercholesterolemia, α-thalassemia and ocular albinism type 1 [15, 23, 24]. Pairwise BLAST analysis of the wild-type locus revealed that the FRAM and AluY elements at the deletion breakpoints have 75% nucleotide identity within a core ∼110-bp sequence. It is possible that homologous pairing between these regions of identity permitted unequal crossing over between chromosomes, leading to generation of the deleted allele. The discovery of at least four, possibly five, French Canadian families sharing a ChAc-associated haplotype indicates that as with many other mendelian disorders, ChAc may show a founder effect in this population. Routine testing for the EX70_EX73del mutation may therefore be an option for rapid diagnosis of ChAc in French Canadian patients with relevant symptoms. Conventional mutation detection strategies involving amplification of single exons will only detect homozygous carriers of this allele. We have demonstrated that the deletion breakpoint PCR described in this study will be a useful tool for identification of heterozygous EX70_EX73del carriers. Contrary to expectations in a family with such a high degree of consanguinity, the affected members of family 4 do not all share the same disease mutations. Two members are homozygous for the splice-site mutation 4242+ 1G>T; and two members are compound heterozygotes for the 4242+1G>T and the EX70_EX73del alleles. This case highlights the caution needed in interpreting linkage analysis data based on assumptions of consanguinity in a pedigree. The 4242+1G>T splice-site mutation has not only been identified in a French Canadian family, but also in siblings of Algerian origin. Analysis of the Algerian patients revealed that they were homozygous for the same 8-SNP haplotype and GATA89a11 and D9S1674 alleles as patients 4-7 and 4-8 (data not shown). Algeria was occupied by France from 1830 to 1962 and as such received French immigration during this time. However, there is no evidence of French or French Canadian ancestry in this Algerian pedigree (I. Sibon 2004, personal communication), so inheritance of a common ancestral allele cannot be confirmed. Interestingly, the EX70_EX73del mutation also spans the terminal two exons of GNA14, which encodes Gα subunit 14. The α subunits of G-proteins form heterotrimeric complexes with β and γ subunits; these complexes are involved in signal transduction from cell surface receptors to a variety of intracellular effectors. GNA14 is positioned just upstream of the gene encoding Gα subunit q, GNAQ; the two genes have probably arisen through tandem duplication [25]. Although GNAQ is expressed in a wide range of tissues, GNA14 mRNA is virtually undetectable in adult tissue and appears to be restricted to foetal lung, kidney and liver, at levels far lower than GNAQ [18]. Except for a possibly higher incidence of seizures

[26], preliminary investigations suggest that the individuals who harbour the deletion of the 3′ termini of VPS13A and GNA14 have no obvious phenotypic differences from those who have mutations affecting VPS13A only. This would imply that either Gα14 function does not depend on the presence of its carboxy-terminal region, or that its absence is fully compensated for by, e.g. other Gα subunit proteins. Indeed, many biochemical activities ascribed to Gα14 have also been observed in other Gα proteins [27–30]. However, detailed clinical analysis of individuals harbouring the GNA14 deletion may reveal subtle differences in phenotype that could give clues to Gα14’s physiological role. Acknowledgements The authors would like to thank the families for their participation, Lorne Lonie for DHPLC analysis and Carlos Singer and Igor Sibon for collection of patient samples and helpful discussions. This work was supported by funds from the Wellcome Trust (Wellcome Trust Prize Studentship 060886/Z/00/Z, held by C. D.-S.). C.D.-S. is currently supported by a European Molecular Biology Organisation postdoctoral fellowship (ref: ALTF166-2004). A. V.-B. is supported by a Marie Curie postdoctoral fellowship (ref: QLGA-CT-2001-51850). A.J. received funding from the Belgische Stichting Roeping/Foundation Belge de la Vocation and is currently receiving a fellowship from the Savoy Foundation for Epilepsy Research. E.A. was funded by a grant from the Canadian Institutes of Health Research. A.P.M. is a Wellcome Trust Principal Research Fellow (ref: 045093/Z/95). The experiments performed in this study comply with current UK laws.

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