Search

Haemophilia published by John Wiley & Sons Ltd Introduction: Advances in genomic sequencing have facilitated the sequencing of genes associated with disorders of haemostasis. The identification of variants within genes and access to curated data incorporating structural, functional, evolutionary as well as phenotypic data has become increasingly important in order to ascribe pathogenicity. Aim: The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Database Project aims to provide a single port of entry to a web-accessible resource for variants in genes involved in clinical bleeding disorders. Results: New databases have evolved from previously developed single gene variant coagulation database projects, incorporating new data, new analysis tools and a new common database architecture with new interfaces and filters. These new databases currently present information about the genotype, phenotype (laboratory and clinical) and structural and functional effects of variants described in the genes of factor (F) VII (F7), FVIII (F8), FIX (F9) and von Willebrand factor (VWF). Conclusion: The project has improved the quality and quantity of information available to the haemostasis research and clinical communities, thereby enabling accurate classification of disease severity in order to make assessments of likely pathogenicity.

Hereditary blood coagulation factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder resulting from variants in the gene encoding FVII (F7). Integration of genetic variation with functional consequences on protein function is essential for the interpretation of the pathogenicity of novel variants. Here, we describe the integration of previous locus‐specific databases for F7 into a single curated database with enhanced features. The database provides access to in silico analyses that may be useful in the prediction of variant pathogenicity as well as cross‐species sequence alignments, structural information, and functional and clinical severity described for each variant, where appropriate. The variant data is shared with the F7 Leiden Open Variation Database. The updated database now includes 221 unique variants, representing gene variants identified in 728 individuals. Single nucleotide variants are the most common type (88%) with missense representing 74% of these variants. A number of variants are found with relatively high minor allele frequencies that are not pathogenic but contribute significantly to the likely pathogenicity of coinherited variants due to their effect on FVII plasma levels. This comprehensive collection of curated information significantly aids the assessment of pathogenicity.

With the advent of large-scale next-generation sequencing initiatives, there is an increasing importance to interpret and understand the potential phenotypic influence of identified genetic variation and its significance in the human genome. Bioinformatics analyses can provide useful information to assist with variant interpretation. This review provides an overview of tools/resources currently available, and how they can help predict the impact of genetic variation at the deoxyribonucleic acid, ribonucleic acid, and protein level.

Introduction: Inherited macrothrombocytopenia represents a heterogeneous group of disorders which are characterised by the presence of a reduced number of abnormally large platelets in the circulation, which may or may not be associated with a bleeding tendency. In spite of several causative genes having been identified, the underlying genetic defects remain to be identified in approximately half of the cases. Aims: To understand the molecular pathology of isolated giant platelet disorder from India. Materials and Methods: We studied 112 cases who were referred for investigation of macrothrombocytopenia. Agonist induced platelet aggregation and platelet GP1b/IX/V receptor expression were investigated to assess GP1b/IX/V receptor expression and the GP1BA, GP1BB, GP9, ABCG5, ABCG8, TUBB1 and MYH9 genes were analysed to identify candidate gene defects. Results: Twenty three candidate gene defects were identified in 48 of 112 cases, 20 of which were novel. Of the candidate defects identified, 91% were missense and 9% were nonsense variations. The missense variations were in GP9 (9), ABCG5 (4), GP1BB (3), GP1BA (3), and MYH9 (2), while the nonsense defects occurred in MYH9 (1) and GP1BA (1). Conclusions: This study increases the understanding of the molecular basis of an isolated giant platelet disorder, a common heterogeneous condition prevalent in North and Eastern India.

VWF variants c.2365A>G and c.2385T>C independently influence VWF biosynthesis and clearance, increasing VWF plasma levels.Commonly inherited VWF variants can directly influence the protein and may contribute to hemostatic and thrombotic disease risk/severity.

SummarySeveral cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however, these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17–18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships.

Inherited bleeding, thrombotic and platelet disorders (BPDs) are diseases affecting approximately 300 individuals per million births. With the exception of haemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialised tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing (HTS) platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants (SNVs), short insertions/deletions (indels) and large copy number variants (CNVs), though not inversions, which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples respectively from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology while the remainder had ana priorihighly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only eight of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5′ of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.

Individuals born of consanguineous union have segments of their genomes that are homozygous as a result of inheriting identical ancestral genomic segments through both parents. One consequence of this is an increased incidence of recessive disease within these sibships. Theoretical calculations predict that 6% (1/16) of the genome of a child of first cousins will be homozygous and that the average homozygous segment will be 20 cM in size. We assessed whether these predictions held true in populations that have preferred consanguineous marriage for many generations. We found that in individuals with a recessive disease whose parents were first cousins, on average, 11% of their genomes were homozygous (n = 38; range 5%-20%), with each individual bearing 20 homozygous segments exceeding 3 cM (n = 38; range of number of homozygous segments 7-32), and that the size of the homozygous segment associated with recessive disease was 26 cM (n = 100; range 5-70 cM). These data imply that prolonged parental inbreeding has led to a background level of homozygosity increased approximately 5% over and above that predicted by simple models of consanguinity. This has important clinical and research implications.

Mutations in the ASPM gene at the MCPH5 locus are expected to be the most common cause of human autosomal recessive primary microcephaly (MCPH), a condition in which there is a failure of normal fetal brain development, resulting in congenital microcephaly and mental retardation. We have performed the first comprehensive mutation screen of the 10.4-kb ASPM gene, identifying all 19 mutations in a cohort of 23 consanguineous families. Mutations occurred throughout the ASPM gene and were all predicted to be protein truncating. Phenotypic variation in the 51 affected individuals occurred in the degree of microcephaly (5-11 SDs below normal) and of mental retardation (mild to severe) but appeared independent of mutation position.

One of the most notable trends in mammalian evolution is the massive increase in size of the cerebral cortex, especially in primates. Humans with autosomal recessive primary microcephaly (MCPH) show a small but otherwise grossly normal cerebral cortex associated with mild to moderate mental retardation. Genes linked to this condition offer potential insights into the development and evolution of the cerebral cortex. Here we show that the most common cause of MCPH is homozygous mutation of ASPM, the human ortholog of the Drosophila melanogaster abnormal spindle gene (asp), which is essential for normal mitotic spindle function in embryonic neuroblasts. The mouse gene Aspm is expressed specifically in the primary sites of prenatal cerebral cortical neurogenesis. Notably, the predicted ASPM proteins encode systematically larger numbers of repeated 'IQ' domains between flies, mice and humans, with the predominant difference between Aspm and ASPM being a single large insertion coding for IQ domains. Our results and evolutionary considerations suggest that brain size is controlled in part through modulation of mitotic spindle activity in neuronal progenitor cells.

Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain.

Microcephaly is the clinical finding of a head circumference measurement greater than three standard deviations (SD) below the population mean for age and sex. It is usually accompanied by mental retardation and there are many diagnoses with both environmental and genetic aetiologies.1 Autosomal recessive primary microcephaly (MCPH) (MIM 251200) is a disorder in which affected subjects are born with a small head circumference, explained by a cerebral cortex of reduced size, and are mentally retarded. The brain is structurally normal and, apart from the intellectual impairment, there are no other significant neurological problems, dysmorphic features, or malformations.2,3 In a study carried out in The Netherlands,4 the incidence of MCPH was approximately 1/250 000 but it is probably greater in populations with a high rate of consanguineous marriages. MCPH has been shown to be genetically heterogeneous with the identification of five loci: MCPH1 on 8p23,5 MCPH2 on 19q13,6 MCPH3 on 9q34,7 MCPH4 on 15q15-q21,8 and MCPH5 on 1q31.9,10 MCPH1, 2, and 3 were mapped in northern Pakistani families, MCPH4 in a Moroccan family, and MCPH5 in northern Pakistani and Turkish families. Here we report the identification by autozygosity mapping11 of a novel locus for primary microcephaly, MCPH6, in a north eastern Brazilian family. ### Key points

Kufor-Rakeb syndrome is an autosomal recessive nigro-striatal-pallidal-pyramidal neurodegeneration. The onset is in the teenage years with clinical features of Parkinson's disease plus spasticity, supranuclear upgaze paresis, and dementia. Brain scans show atrophy of the globus pallidus and pyramids and, later, widespread cerebral atrophy. We report linkage in Kufor-Rakeb syndrome to a 9 cM region of chromosome 1p36 delineated by the markers D1S436 and D1S2843, with a maximum multipoint lod score of 3.6. Keywords: Kufor-Rakeb syndrome; autozygosity mapping; Parkinson's disease; chromosome 1p36

The primary cilium is an antenna-like structure that protrudes from the cell surface of quiescent/differentiated cells and participates in extracellular signal processing. Here, we report that mice deficient for the lipid 5-phosphatase Inpp5e develop a multiorgan disorder associated with structural defects of the primary cilium. In ciliated mouse embryonic fibroblasts, Inpp5e is concentrated in the axoneme of the primary cilium. Inpp5e inactivation did not impair ciliary assembly but altered the stability of pre-established cilia after serum addition. Blocking phosphoinositide 3-kinase (PI3K) activity or ciliary platelet-derived growth factor receptor alpha (PDGFRalpha) restored ciliary stability. In human INPP5E, we identified a mutation affecting INPP5E ciliary localization and cilium stability in a family with MORM syndrome, a condition related to Bardet-Biedl syndrome. Together, our results show that INPP5E plays an essential role in the primary cilium by controlling ciliary growth factor and PI3K signaling and stability, and highlight the consequences of INPP5E dysfunction., Journal Article, Research Support, N.I.H. Extramural, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Background: Autosomal recessive primary microcephaly (MCPH) is a model disease to study human neurogenesis. In affected individuals the brain grows at a reduced rate during fetal life resulting in a small but structurally normal brain and mental retardation. The condition is genetically heterogeneous with mutations in ASPM being most commonly reported. Methods and results: We have examined this further by studying three cohorts of microcephalic children to extend both the phenotype and the mutation spectrum. Firstly, in 99 consecutively ascertained consanguineous families with a strict diagnosis of MCPH, 41 (41%) were homozygous at the MCPH5 locus and all but two families had mutations. Thus, 39% of consanguineous MCPH families had homozygous ASPM mutations. Secondly, in 27 non-consanguineous, predominantly Caucasian families with a strict diagnosis of MCPH, 11 (40%) had ASPM mutations. Thirdly, in 45 families with a less restricted phenotype including microcephaly and mental retardation, but regardless of other neurological features, only 3 (7%) had an ASPM mutation. This report contains 27 novel mutations and almost doubles the number of MCPH associated ASPM mutations known to 57. All but one of the mutations lead to the use of a premature termination codon, 23 were nonsense mutations, 28 deletions or insertions, 5 splicing, and 1 was a translocation. Seventeen of the 57 mutations were recurrent. There were no definitive missense mutations found nor was there any mutation/phenotype correlation. ASPM mutations were found in all ethnic groups studied. Conclusion: This study confirms that mutations in ASPM are the most common cause of MCPH, that ASPM mutations are restricted to individuals with an MCPH phenotype, and that ASPM testing in primary microcephaly is clinically useful.

A consanguineous pedigree is described where 14 individuals are affected with a novel autosomal recessive disorder, which causes static moderate mental retardation, truncal obesity, a congenital nonprogressive retinal dystrophy and micropenis in males. We have tentatively named this condition MORM syndrome. It shows similarities to Bardet-Biedl syndrome and Cohen syndrome, but can be distinguished by clinical features; the age of onset and nonprogressive nature of the visual impairment, the lack of characteristic facies, skin or gingival infection, microcephaly, 'mottled retina', polydactyly and small penis without testicular anomalies. Furthermore, linkage to the known Bardet-Biedl (BBS1-8) and Cohen syndrome loci was excluded. Autozygosity mapping identified a single homozygous subtelomeric region shared by all affecteds on chromosome 9q34.3, with a maximum LOD score of 5.64. We believe this to be the first example of the identification of a subtelomeric recessive locus by autozygosity mapping.

Introduction: In recent years, the role of copy number variation (CNV) in the pathogenesis of type 1 von Willebrand disease (VWD) has been extensively explored. Large cohort studies including the MCMDM-1VWD and French studies have identified a total of eight partial VWF gene deletions, involving single and multiple exons, as well as one duplication. Furthermore, breakpoints have been identified within the MCMDM-1VWD cohort study for deletions involving exons 3, 32-34 and 33-34. Aim: To characterize and determine heterozygous deletion breakpoints of a deletion within the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) cohort, previously identified through array CGH analysis and to determine the pathogenesis of the deletion through in vitro analysis. Methods: Identification of deletion breakpoints utilized a deletion-specific multiplex PCR approach, whereby two forward primers and one reverse primer were designed to determine the heterozygous exon deletion, based on previous optimization within the MCMDM-1VWD cohort. VWF antigen (VWF:Ag), VWF propeptide (VWFpp), and multimer distribution were performed in a central laboratory. Recombinant exon 33-34 deletion mutant (rVWFdel33-34) was created using site directed mutagenesis. Recombinant vectors were transiently transfected into HEK293T cells and Renilla used as a transfection control. Conditioned media and cellular lysates were collected 48hr post-transfection and analysed by VWF:Ag ELISA, comparing to wild-type VWF (rVWFwt) expression. Results: Three families enrolled in the ZPMCB-VWD displayed a deletion of VWF exon 33-34. The deletion was determined to be 3.4kb in size and led to an in-frame loss of exons 33-34, with breakpoints being identical to those identified in the MCMDM-1VWD study (c.5620+872_5842+2440delinsGCAGCATAAGCATAAAGC). VWFpp/VWF:Ag ratios were elevated in all affected family members with the exon 33-34 deletion (mean 4.3) as opposed to unaffected family members (mean 1.3). These results are consistent with findings in the MCMDM-1VWD family (mean VWFpp/VWF:Ag of 4.8), suggesting increased clearance of VWF. In silico analysis of the breakpoint region suggested no significant repetitive elements and microhomology mediated end-joining was hypothesised as the potential mechanism leading to the deletion. Expression of recombinant mutant rVWFdel33-34 revealed that secretion reduced by 52% in the homozygous state (p Conclusion: Analysis of the exon 33-34 deletion breakpoint revealed no highly homologous repetitive sequences but there was a DNA insertion of 18 bp at the deletion breakpoint. In vitro expression of the exon 33-34 deletion suggests that there is a moderate effect on VWF secretion into conditioned media, without increased intracellular retention. The exon 33-34 deletion leads to the loss of nine cysteine residues. Inter- and intra- disulphide bonding between cysteine residues within VWF monomers and multimers is known to be functionally important in facilitating correct VWF structural orientation and folding within D4-CK domains. Loss of these cysteines may explain the moderate reduction in VWF secretion and potentially reduced plasma survival of the variant VWF. Disclosures Montgomery: Immucor: Patents & Royalties.

Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of the Rab3 pathway implicated in exocytic release of neurotransmitters and hormones, in 12 families with Micro syndrome. We hypothesize that the underlying pathogenesis of Micro syndrome is a failure of exocytic release of ocular and neurodevelopmental trophic factors.

Dear Editor, Kasatkar and colleagues recently reported that the previously described von Willebrand factor (VWF) variant c.6187C>T (p.P2063S) appears to be a common cause of type 3 von Willebrand disease (VWD3) in the Indian population [1]. In support of their conclusion, p.P2063S was the only VWF variant observed in 11 Indian VWD3 index cases (IC), tracked with disease phenotype and was predicted to be deleterious via in silico protein prediction analysis. In order to ensure accurate molecular characterisation of VWF variants, it is essential to consider all available information. Regarding p.P2063S, there are several lines of evidence that contradict the conclusion drawn. p.P2063S is also observed in healthy controls with reported population frequencies between 0.6–2.5% in African-Americans / Caucasians [2–4] and (as reported by Kasatkar and colleagues [1]) 3% in Indians. Given a frequency of 3% in the general population, Hardy-Weinberg equilibrium predicts homozygosity for this variant in approximately 1/1100 individuals. This would suggest a high population frequency of VWD3 if this variant were causative. It is also notable that p.P2063S has not been reported as causal in previous studies of VWD3 in Indians [5–7]. The majority of missense mutations reported in VWD3 patients occur in the VWF propeptide and C-terminus where they are reported to affect protein folding and multimerisation resulting in intracellular retention of the protein [2,7,8]. Kasatkar and colleagues argue that diagnosis of VWD3 relies on defective release or synthesis of VWF [1], however given that p.P2063S has no effect on VWF expression in vitro [2] it seems unlikely that this variant would affect VWF in vivo. This is supported by observations of p.P2063S in VWD patients inherited together with other VWF variants that more readily explain their VWD phenotype (Table 1), most notably in a Turkish VWD3 IC [8]. Table 1 Co-inheritance of p.P2063S and actual VWD causing variants In previous Indian VWD3 studies [5–7], molecular analysis failed to fully account for the genetic cause in 8/54 IC (15%). It is therefore likely that the actual disease causing mutation has yet to be identified by Kasatkar and colleagues, who imply that DNA scanning methods were used to identify mutations in their patient cohort [1]. DNA scanning methods are not 100% sensitive to all sequence variations so may fail to detect the ‘true’ mutation, for example chemical cleavage mismatch analysis identified a homozygous p.P2063S variant in a Turkish VWD3 IC but failed to identify the actual p.Q1734* causative mutation (Table 1) [8]. Alternatively, the causative mutation may have been overlooked if, for example, it were a synonymous variant that disrupts normal splicing or protein folding, or a deep intronic variant causing disruption of splicing outside the VWF regions analysed [9]. In summary, review of the available information supports the conclusion that p.P2063S is a common neutral VWF polymorphic variant. Further studies (for example using mRNA as the template investigated) may identify the underlying cause of VWD in this interesting patient group.

We report a Jordanian Arab family where two sibs developed the classical clinical and radiological features of pantothenate kinase associated neurodegeneration (PKAN, formerly known as Hallervorden-Spatz disease) but in addition had an early onset cerebellar ataxia.1,2 Using polymorphic microsatellite markers we have shown that this family is not linked to the pantothenate kinase gene ( PANK2 ) on chromosome 20.2 We hypothesise that the disorder, Karak syndrome, is novel and a member of the growing family of neurological diseases involving excess cerebral iron accumulation, for example, PKAN, neuroferritinopathy, aceruloplasminaemia, and Friedreich’s ataxia.2–6 Both affected members (fig 1, IV.1 and IV.2) were the product of a normal pregnancy and birth and had normal developmental milestones and progress at school until disease onset at the age of 6 years. They developed an ataxic gait that was slowly progressive, and was associated with decreased school performance. At 8 years of age they developed inverted feet (calcaneovarus), which was associated with frequent falls. Around the age of 9 years, both started to have choreiform movements of all four limbs, more marked in the upper limbs than in the lower limbs. By the age of 10 years the condition had progressed and they were unable to walk without assistance, and they left school soon after. Their mother found difficulty in feeding them because of swallowing problems after the age of 10 years and they were unable to dress, bathe, or feed themselves by their mid-teens. There were no visual or auditory symptoms or history of epilepsy. Both parents and four older sibs, two brothers and two sisters, were in good health. The parents were first cousins and came from an inbred family (fig 1). The family lived in Karak, a town in southern Jordan. Figure 1 A simplified pedigree of the research family. Affected …

Abstract 2218 Introduction: Type 1 von Willebrand disease (VWD) results from a partial quantitative deficiency of von Willebrand factor (VWF). Three multicentre studies were recently conducted to elucidate the molecular and clinical features of type 1 VWD; undertaken in the European Union (EU), Canada and the UK. All three successfully identified genetic alterations likely to cause type 1 VWD, but failed to identify a genetic cause in approximately 30–40% of patients. To date, relatively little is known regarding the extent to which large-scale deletions or duplications of VWF contribute to the pathogenesis of type 1 VWD due to the difficulty in identifying such events in heterozygous individuals. The aim of this initial investigation, on behalf of the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) and the EU study on VWD (EU-VWD), was to ascertain whether copy number variation (CNV) within VWF contributes to type 1 VWD phenotype in a cohort of index cases (IC) recruited by the EU study. Methods: Twenty-five mutation-negative IC and a further seven IC, in which the identified mutation did not explain the VWD phenotype, were investigated. Multiplex ligation-dependent probe amplification (MLPA), a well established technique based on sequence-specific probe hybridization to genomic DNA, was used to screen for CNV within VWF, utilizing a recently released VWF MLPA kit (P011-A1/P012-A1; MRC-Holland). In addition, five healthy control individuals (HC) were also screened. Fragment size analysis on an ABI 3730 DNA sequencer was followed by calculation of copy number ratios for each exon-specific amplicon in comparison with HC. Results: Analysis highlighted heterozygous CNV in 7/32 IC; five deletions and two apparent duplications. Regarding the deletions; three were the recently described in-frame deletion of exons 4 and 5 (p.Asp75_Gly178del; Sutherland et al., 2009), the other two patients both had a novel deletion of exons 32–34. Interestingly, the exon 32–34 deletion was also predicted to be in-frame (p.Arg1819_Cys1948delinsSer) and in both IC was associated with abnormal multimers (AbM) and low VWF:Ag (32 and 12 IU/dL respectively) and VWF:RCo (23 and 11 IU/dL respectively) levels. Deletions in all five cases segregated with disease phenotype. The two apparent duplications of exon 2 were both shown to be false positive results following further investigation with a new version of the MLPA kit (P011-B1/P012-B1). Conclusions: CNV of VWF has been shown to contribute to the pathogenesis of type 1 VWD. MLPA analysis of VWF has identified a previously described and a novel heterozygous deletion, both of which result in in-frame deletion of the VWF protein. The analysis also demonstrates that care must be taken when analyzing MLPA data to avoid false positive results. This adds to the mutations detected in the EU cohort, bringing the number to 113/150 IC (75%). Furthermore, MLPA analysis has increased the overall mutation detection rate in EU IC with AbM to 100%. These findings suggest that CNV is a significant contributor to mutation spectrum in type 1 VWD. Application of MLPA to the wider ZPMCB-VWD and EU-VWD patient cohorts will enable ascertainment of the contribution of large deletion/duplication events to all types of VWD pathogenesis. Disclosures: Vijzelaar: MRC-Holland b.v.: Employment.

Nat. Genet. 37, 353–355 (2005). The name of the fourth author was incorrect. The correct name is Sofia B. Lizarraga.

Background and objectives: Locus heterogeneity is well established in autosomal recessive primary microcephaly (MCPH) and to date five loci have been mapped. However, the relative contributions of these loci have not been assessed and genotype-phenotype correlations have not been investigated. Design: A study population of 56 consanguineous families resident in or originating from northern Pakistan was ascertained and assessed by the authors. A panel of microsatellite markers spanning each of the MCPH loci was designed, against which the families were genotyped. Results: The head circumference of the 131 affected subjects ranged from 4 to 14 SD below the mean, but there was little intrafamilial variation among affecteds (± 1 SD). MCPH5 was the most prevalent, with 24/56 families consistent with linkage; 2/56 families were compatible with linkage to MCPH1, 10/56 to MCPH2, 2/56 to MCPH3, none to MCPH4, and 18/56 did not segregate with any of the loci. Conclusions: MCPH5 is the most common locus in this population. On clinical grounds alone, the phenotype of families linked to each MCPH locus could not be distinguished. We have also shown that further MCPH loci await discovery with a number of families as yet unlinked.