CD3G gene defects in familial autoimmune thyroiditis.

The patients with CD3γ deficiency can present with different clinical findings despite having the same homozygous mutation. We report three new CD3gamma-deficient siblings from a consanguineous family with a combined T-B+NK+ immunodeficiency and their variable clinical and cellular phenotypes despite the same homozygous mutation of the CD3G gene (c.80-1G>C). We also re-evaluate a previously reported non-consanguineous family with two CD3gamma-deficient siblings with the same mutation. The median age at diagnosis was 11 years (14 months-20 years). We found all five patients to display autoimmunity: autoimmune thyroiditis (n = 5), autoimmune haemolytic anaemia (n = 2), immune thrombocytopenia (n = 1), autoimmune hepatitis (n = 1), minimal change nephrotic syndrome (n = 1), vitiligo (n = 1) and positive antinuclear antibodies (n = 3) as well as high IgE (n = 2) and atopic eczema (n = 2). While CD3(+) TCRαß+T cell percentages were low in all patients, only one had lymphopenia and 3 had CD3(+) T cell lymphopenia. Strikingly, we report frequent and multiple autoimmunity in tested heterozygous carriers in both families (n = 6; in 67%), and frequent autoimmunity in family members not available for testing (n = 5, in 80%). The results suggest that CD3G should be studied as a candidate gene for autoimmunity and that CD3gamma deficiency should be considered among other primary immunodeficiencies with predominantly autoimmune manifestations.

Localisation of the Fanconi anaemia complementation group A gene to chromosome 16q24.3.

Fanconi anaemia (FA) is an autosomal recessive disorder associated with diverse developmental abnormalities, bone-marrow failure and predisposition to cancer. FA cells show increased chromosome breakage and hypersensitivity to DNA cross-linking agents such as diepoxybutane and mitomycin C. Somatic-cell hybridisation analysis of FA cell lines has demonstrated the existence of at least five complementation groups (FA-A to FA-E), the most common of which is FA-A. This genetic heterogeneity has been a major obstacle to the positional cloning of FA genes by classical linkage analysis. The FAC gene was cloned by functional complementation, and localised to chromosome 9q22.3 (ref. 2), but this approach has thus far failed to yield the genes for the other complementation groups. We have established a panel of families classified as FA-A by complementation analysis, and used them to search for the FAA gene by linkage analysis. We excluded the previous assignment by linkage of an FA gene to chromosome 20q, and obtained conclusive evidence for linkage of FAA to microsatellite markers on chromosome 16q24.3. Strong evidence of allelic association with the disease was detected with the marker D16S303 in the Afrikaner population of South Africa, indicating the presence of a founder effect.

Spontaneous functional correction of homozygous fanconi anaemia alleles reveals novel mechanistic basis for reverse mosaicism.

Somatic mosaicism due to reversion of a pathogenic allele to wild type has been described in several autosomal recessive disorders. The best known mechanism involves intragenic mitotic recombination or gene conversion in compound heterozygous patients, whereby one allele serves to restore the wild-type sequence in the other. Here we document for the first time functional correction of a pathogenic microdeletion, microinsertion and missense mutation in homozygous Fanconi anaemia (FA) patients resulting from compensatory secondary sequence alterations in cis. The frameshift mutation 1615delG in FANCA was compensated by two additional single base-pair deletions (1637delA and 1641delT); another FANCA frameshift mutation, 3559insG, was compensated by 3580insCGCTG; and a missense mutation in FANCC(1749T-->G, Leu496Arg) was altered by 1748C-->T, creating a cysteine codon. Although in all three cases the predicted proteins were different from wild type, their cDNAs complemented the characteristic hypersensitivity of FA cells to crosslinking agents, thus establishing a functional correction to wild type.

Promoter mutation is a common variant in GJC2-associated Pelizaeus-Merzbacher-like disease.

Pelizaeus-Merzbacher-like disease (PMLD) is a clinically and genetically heterogeneous neurological disorder of cerebral hypomyelination. It is clinically characterised by early onset (usually infantile) nystagmus, impaired motor development, ataxia, choreoathetoid movements, dysarthria and progressive limb spasticity. We undertook autozygosity mapping studies in a large consanguineous family of Pakistani origin in which affected children had progressive lower limb spasticity and features of cerebral hypomyelination on MR brain imaging. SNP microarray and microsatellite marker analysis demonstrated linkage to chromosome 1q42.13-1q42.2. Direct sequencing of the gap junction protein gamma-2 gene, GJC2, identified a promoter region mutation (c.-167A>G) in the non-coding exon 1. The c.-167A>G promoter mutation was identified in a further 4 individuals from two families (who were also of Pakistani origin) with clinical and radiological features of PMLD in whom previous routine diagnostic screening of GJC2 had been reported as negative. A common haplotype was identified at the GJC2 locus in the three mutation-positive families, consistent with a common origin for the mutation and likely founder effect. This promoter mutation has only recently been reported in GJC2-PMLD but it has been postulated to affect the binding of the transcription factor SOX10 and appears to be a prevalent mutation, accounting for ~29% of reported patients with GJC2-PMLD. We propose that diagnostic screening of GJC2 should include sequence analysis of the non-coding exon 1, as well as the coding regions to avoid misdiagnosis or diagnostic delay in suspected PMLD.

Germline mutation in DOK7 associated with fetal akinesia deformation sequence.

BACKGROUND: Fetal akinesia deformation sequence syndrome (FADS) is a heterogeneous disorder characterised by fetal akinesia and developmental defects including, in some case, pterygia. Multiple pterygium syndromes (MPS) are traditionally divided into prenatally lethal and non-lethal (such as Escobar) types. Previously, we and others reported that homozygous mutations in the fetal acetylcholine receptor gamma subunit (CHRNG) can cause both lethal and non-lethal MPS, demonstrating that pterygia resulted from fetal akinesia, and that mutations in the acetylcholine receptor subunits CHRNA1, CHRND, and Rapsyn (RAPSN) can also result in a MPS/FADS phenotype. METHODS: We hypothesised that mutations in other acetylcholine receptor related genes may interfere with neurotransmission at the neuromuscular junction and so we analysed 14 cases of lethal MPS/FADS without CHRNG, CHRNA1, CHRNB1, CHRND, or RAPSN mutations for mutations in DOK7. RESULTS: A homozygous DOK7 splice site mutation, c.331+1G>T, was identified in a family with three children affected with lethal FADS. Previously DOK7 mutations have been reported to underlie a congenital myaesthenic syndrome with a characteristic "limb girdle" pattern of muscle weakness. CONCLUSION: This finding is consistent with the hypothesis that whereas incomplete loss of DOK7 function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype.

Comparison of multiple electrode aggregometry with lumi-aggregometry for the diagnosis of patients with mild bleeding disorders.

Essentials There is a clinical need for new technologies to measure platelet function in whole blood. Mild bleeding disorders were evaluated using multiple electrode aggregometry (MEA). MEA is insensitive at detecting patients with mild platelet function and secretion defects. More studies are required to investigate MEA in patients with a defined set of platelet disorders. SUMMARY: Background Multiple electrode aggregometry (MEA) measures changes in electrical impedance caused by platelet aggregation in whole blood. This approach is faster, more convenient and offers the advantage over light transmission aggregometry (LTA) of assessing platelet function in whole blood and reducing preanalytical errors associated with preparation of platelet-rich plasma (PRP). Several studies indicate the utility of this method in assessing platelet inhibition in individuals taking antiplatelet agents (e.g. aspirin and clopidogrel). Objective Our current study sought to evaluate the ability of MEA in diagnosing patients with mild bleeding disorders by comparison with light transmission lumi-aggregometry (lumi-LTA). Methods Forty healthy subjects and 109 patients with a clinical diagnosis of a mild bleeding disorder were recruited into the UK Genotyping and Phenotyping of Platelets study (GAPP, ISRCTN 77951167). MEA was performed on whole blood using one or two concentrations of ADP, PAR-1 peptide, arachidonic acid and collagen. Lumi-LTA was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Results Of 109 patients tested, 54 (49%) patients gave abnormal responses by lumi-LTA to one or more agonists. In contrast, only 16 (15%) patients were shown to have abnormal responses to one or more agonists by MEA. Conclusions In this study we showed that MEA is less sensitive in identifying patients with abnormal platelet function relative to lumi-LTA.

Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma.

Renal cell carcinoma (RCC), the most common adult kidney neoplasm, is histopathologically heterogeneous, with most sporadic RCCs ( approximately 80%) classified as clear cell (CC) tumors. Chromosome 3p allele loss is the most frequent genetic alteration in RCC but is associated specifically with sporadic and hereditary forms of clear cell RCC (CC-RCC) and is not a feature of non-CC-RCC, such as papillary (chromophilic) RCC. The VHL tumor suppressor gene (TSG) maps to chromosome 3p25, and somatic inactivation of the VHL gene occurs in up to 70% of CC-RCC tumors and cell lines. However, VHL inactivation is not sufficient for CC-RCC tumorigenesis, and inactivation of 3p12-p21 TSG(s) appears to be necessary in CC-RCC irrespective of VHL gene inactivation status. Recently, we demonstrated that the candidate 3p21 TSG, RASSF1A, is hypermethylated in most small cell lung cancers. We have now investigated the role of RASSF1A inactivation in primary RCC tumors. RASSF1A promoter methylation was detected in 23% (32 of 138) of primary CC-RCC tumors. In CC-RCC cell lines, RASSF1A methylation was associated with silencing of RASSF1A expression and restoration of expression after treatment with 5'-azacytidine. The frequency of RASSF1A methylation was similar in CC-RCC with and without VHL gene inactivation (24% versus 21%), and there was no association between epigenetic silencing of the RASSF1A and VHL TSGs, because 0 of 6 tumors with VHL hypermethylation had RASSF1A methylation, and VHL was not methylated in 26 CC-RCCs with RASSF1A methylation. Although 3p allele loss has been reported rarely in papillary RCC, we identified RASSF1A methylation in 44% (12 of 27) of papillary RCCs analyzed. Thus: (a) inactivation of RASSF1A is a frequent event in both CC-RCC and papillary RCC tumors; (b) there is no relationship between epigenetic silencing of RASSF1A and VHL inactivation status in CC-RCC. Fifty-four CC-RCCs analyzed for RASSF1A methylation were informative for 3p21 allele loss, and 20% (7 of 35) with 3p21 allele loss demonstrated RASSF1A methylation. All informative CC-RCCs with 3p21 allele loss and no RASSF1A methylation also demonstrated allele losses at other regions of 3p so that tumorigenesis in these cases may result from: (a) haploinsufficiency of RASSF1A; (b) inactivation of other 3p21 TSGs; or (c) inactivation of 3p TSGs from outside of 3p21. RASSF1A is the first TSG to be inactivated frequently in both papillary and CC-RCCs. The finding of frequent epigenetic inactivation of RASSF1A in papillary RCCs despite previous studies reporting infrequent 3p21 allele loss in this tumor type illustrates how the systematic identification of all major human cancer genes will require detailed analysis of the cancer genome and epigenome.

Loss of heterozygosity mapping at chromosome arm 16q in 712 breast tumors reveals factors that influence delineation of candidate regions.

Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.

Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1alpha in clear cell renal carcinomas.

The transcription factor hypoxia-inducible factor (HIF)-1 is an important mediator of hypoxic adaptation of tumor cells and controls several genes that have been implicated in tumor growth. Oxygen-dependent degradation of HIF-1alpha, the regulatory subunit, requires binding to the von Hippel Lindau (VHL) protein. Because functional inactivation of the VHL tumor suppressor gene occurs in up to 70% of clear cell renal carcinomas, we investigated whether this results in overexpression of HIF-1alpha and its target genes. Immunoblotting revealed increased expression of HIF-1alpha in 24 of 32 (75%) clear cell renal carcinomas but only 3 of 8 non-clear cell renal tumors. Somatic mutations of the VHL gene were detected only in clear cell renal carcinomas that overexpressed HIF-1alpha. None of the HIF-1alpha-negative tumors displayed a VHL mutation. The level of HIF-1alpha mRNA was not different between tumors and adjacent kidney tissue. Immunohistochemistry revealed distinct patterns of nuclear staining for HIF-1alpha, depending on histological type and overall abundance of HIF-1alpha. In those clear cell renal carcinomas that showed increased expression on immunoblots, HIF-1alpha was expressed in almost all cells. In the remaining clear cell and in non-clear cell tumors, staining was focal; these different patterns thus were compatible with genetic stabilization in contrast to microenvironmental stimulation of HIF-1alpha as the primary mechanism. The mRNA expression of two known target genes of HIF-1alpha, vascular endothelial growth factor and glucose transporter 1, increased progressively with increasing amounts of HIF-1alpha in tumor extracts. In addition, glucose transporter 1 protein levels correlated with HIF-1alpha abundance. In conclusion, the data provide in vivo evidence for a constitutive up-regulation of HIF-1alpha in the majority of clear cell renal carcinomas, which leads to more widespread accumulation of this transcription factor than hypoxic stimulation. These observations are most likely linked to functional inactivation of the VHL gene product. Increased expression of HIF-1alpha is associated with alterations in gene expression patterns that are likely to contribute to tumor phenotype and progression.

Deletion and reduced expression of the Fanconi anemia FANCA gene in sporadic acute myeloid leukemia.

Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder caused by mutations in one of seven known genes (FANCA,C,D2,E,F,G and BRCA2). Mutations in the FANCA gene are the most prevalent, accounting for two-thirds of FA cases. Affected individuals have greatly increased risks of acute myeloid leukemia (AML). This raises the question as to whether inherited or acquired mutations in FA genes might be involved in the development of sporadic AML. Quantitative fluorescent PCR was used to screen archival DNA from sporadic AML cases for FANCA deletions, which account for 40% of FANCA mutations in FA homozygotes. Four heterozygous deletions were found in 101 samples screened, which is 35-fold higher than the expected population frequency for germline FANCA deletions (P<0.0001). Sequencing FANCA in the AML samples with FANCA deletions did not detect mutations in the second allele and there was no evidence of epigenetic silencing by hypermethylation. However, real-time quantitative PCR analysis in these samples showed reduced expression of FANCA compared to nondeleted AML samples and to controls. These findings suggest that gene deletions and reduced expression of FANCA may be involved in the promotion of genetic instability in a subset of cases of sporadic AML.

A locus for asphyxiating thoracic dystrophy, ATD, maps to chromosome 15q13.

Asphyxiating thoracic dystrophy (ATD), or Jeune syndrome, is a multisystem autosomal recessive disorder associated with a characteristic skeletal dysplasia and variable renal, hepatic, pancreatic, and retinal abnormalities. We have performed a genome wide linkage search using autozygosity mapping in a cohort of four consanguineous families with ATD, three of which originate from Pakistan, and one from southern Italy. In these families, as well as in a fifth consanguineous family from France, we localised a novel ATD locus (ATD) to chromosome 15q13, with a maximum cumulative two point lod score at D15S1031 (Zmax=3.77 at theta=0.00). Five consanguineous families shared a 1.2 cM region of homozygosity between D15S165 and D15S1010. Investigation of a further four European kindreds, with no known parental consanguinity, showed evidence of marker homozygosity across a similar interval. Families with both mild and severe forms of ATD mapped to 15q13, but mutation analysis of two candidate genes, GREMLIN and FORMIN, did not show pathogenic mutations.

Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family.

BACKGROUND: Primary pulmonary hypertension (PPH), resulting from occlusion of small pulmonary arteries, is a devastating condition. Mutations of the bone morphogenetic protein receptor type II gene (BMPR2), a component of the transforming growth factor beta (TGF-beta) family which plays a key role in cell growth, have recently been identified as causing familial PPH. We have searched for BMPR2 gene mutations in sporadic PPH patients to determine whether the same genetic defect underlies the more common form of the disorder. METHODS: We investigated 50 unrelated patients, with a clinical diagnosis of PPH and no identifiable family history of pulmonary hypertension, by direct sequencing of the entire coding region and intron/exon boundaries of the BMPR2 gene. DNA from available parent pairs (n=5) was used to assess the occurrence of spontaneous (de novo) mutations contributing to sporadic PPH. RESULTS: We found a total of 11 different heterozygous germline mutations of the BMPR2 gene in 13 of the 50 PPH patients studied, including missense (n=3), nonsense (n=3), and frameshift (n=5) mutations each predicted to alter the cell signalling response to specific ligands. Parental analysis showed three occurrences of paternal transmission and two of de novo mutation of the BMPR2 gene in sporadic PPH. CONCLUSION: The sporadic form of PPH is associated with germline mutations of the gene encoding the receptor protein BMPR-II in at least 26% of cases. A molecular classification of PPH, based upon the presence or absence of BMPR2 mutations, has important implications for patient management and screening of relatives.

Use of next-generation sequencing and candidate gene analysis to identify underlying defects in patients with inherited platelet function disorders.

BACKGROUND: Inherited platelet function disorders (PFDs) are heterogeneous, and identification of the underlying genetic defects is difficult when based solely on phenotypic and clinical features of the patient. OBJECTIVE: To analyze 329 genes regulating platelet function, number, and size in order to identify candidate gene defects in patients with PFDs. PATIENTS/METHODS: Targeted analysis of candidate PFD genes was undertaken after next-generation sequencing of exomic DNA from 18 unrelated index cases with PFDs who were recruited into the UK Genotyping and Phenotyping of Platelets (GAPP) study and diagnosed with platelet abnormalities affecting either Gi signaling (n = 12) or secretion (n = 6). The potential pathogenicity of candidate gene defects was assessed using computational predictive algorithms. RESULTS: Analysis of the 329 candidate PFD genes identified 63 candidate defects, affecting 40 genes, among index cases with Gi signaling abnormalities, while 53 defects, within 49 genes, were identified among patients with secretion abnormalities. Homozygous gene defects were more commonly associated with secretion abnormalities. Functional annotation analysis identified distinct gene clusters in the two patient subgroups. Thirteen genes with significant annotation enrichment for 'intracellular signaling' harbored 16 of the candidate gene defects identified in nine index cases with Gi signaling abnormalities. Four gene clusters, representing 14 genes, with significantly associated gene ontology annotations were identified among the cases with secretion abnormalities, the most significant association being with 'establishment of protein localization.' CONCLUSION: Our findings demonstrate the genetic complexity of PFDs and highlight plausible candidate genes for targeted analysis in patients with platelet secretion and Gi signaling abnormalities.

Spectrum of mutations in the Fanconi anaemia group G gene, FANCG/XRCC9.

FANCG was the third Faconi anaemia gene identified and proved to be identical to the previously cloned XRCC9 gene. We present the pathogenic mutations and sequence variants we have so far identified in a panel of FA-G patients. Mutation screening was performed by PCR, single strand conformational polymorphism analysis and protein truncation tests. Altogether 18 mutations have been determined in 20 families - 97% of all expected mutant alleles. All mutation types have been found, with the exception of large deletions, the large majority is predicted to lead to shortened proteins. One stop codon mutation, E105X, has been found in several German patients and this founder mutation accounts for 44% of the mutant FANCG alleles in German FA-G patients. Comparison of clinical phenotypes shows that patients homozygous for this mutation have an earlier onset of the haematological disorder than most other FA-G patients. The mouse Fancg sequence was established in order to evaluate missense mutations. A putative missense mutation, L71P, in a possible leucine zipper motif may affect FANCG binding of FANCA and seems to be associated with a milder clinical phenotype.

Heterogeneous spectrum of mutations in the Fanconi anaemia group A gene.

Fanconi anaemia (FA) is a genetically heterogeneous autosomal recessive disorder associated with chromosomal fragility, bone-marrow failure, congenital abnormalities and cancer. The gene for complementation group A (FAA), which accounts for 60-65% of all cases, has been cloned, and is composed of an open reading frame of 4.3 kb, which is distributed among 43 exons. We have investigated the molecular pathology of FA by screening the FAA gene for mutations in a panel of 90 patients identified by the European FA research group, EUFAR. A highly heterogeneous spectrum of mutations was identified, with 31 different mutations being detected in 34 patients. The mutations were scattered throughout the gene, and most are likely to result in the absence of the FAA protein. A surprisingly high frequency of intragenic deletions was detected, which removed between 1 and 30 exons from the gene. Most microdeletions and insertions occurred at homopolymeric tracts or direct repeats within the coding sequence. These features have not been observed in the other FA gene which has been cloned to date (FAC) and may be indicative of a higher mutation rate in FAA. This would explain why FA group A is much more common than the other complementation groups. The heterogeneity of the mutation spectrum and the frequency of intragenic deletions present a considerable challenge for the molecular diagnosis of FA. A scan of the entire coding sequence of the FAA gene may be required to detect the causative mutations, and scanning protocols will have to include methods which will detect the deletions in compound heterozygotes.

Molecular genetic analysis of FIH-1, FH, and SDHB candidate tumour suppressor genes in renal cell carcinoma.

BACKGROUND: Overexpression of the hypoxia inducible factor 1 (HIF-1) and HIF-2 transcription factors and the consequent upregulation of hypoxia inducible mRNAs is a feature of many human cancers and may be unrelated to tissue hypoxia. Thus, the VHL (von Hippel-Lindau) tumour suppressor gene (TSG) regulates HIF-1 and HIF-2 expression in normoxia by targeting the alpha subunits for ubiquitination and proteolysis. Inactivation of the VHL TSG in VHL tumours and in sporadic clear cell renal cell carcinoma (RCC) results in overexpression of HIF-1 and HIF-2. However, RCC without VHL inactivation may demonstrate HIF upregulation, suggesting that VHL independent pathways for HIF activation also exist. In RCC, three candidate HIF activating genes exist-FIH-1 (factor inhibiting HIF), SDHB, and FH-which may be dependent or independent of VHL inactivation. AIMS: To investigate FIH-1, SDHB, and FH for somatic mutations in sporadic RCC. METHODS: Gene mutation was analysed in primary RCCs (clear cell RCCs, papillary RCCs, and oncocytomas) and RCC cell lines. SDHB mutation analysis was performed by denaturing high performance liquid chromatography followed by direct sequencing of aberrant PCR products. FH and FIH-1 mutation analysis were performed by single stranded conformational polymorphism and direct sequencing of PCR products. RESULTS: No mutations were identified in the three genes investigated. CONCLUSIONS: There was no evidence to suggest that somatic mutations occur in the FH, FIH-1, or SDHB TSGs in sporadic RCCs.

Genotyping and phenotyping of platelet function disorders.

The majority of patients with platelet function disorders (PFDs) have normal platelet counts and mild day-to-day bleeding symptoms, but are at risk of major hemorrhage at times of trauma, surgery, or childbirth. This group is challenging to investigate, because the assays are often time-intensive and labour-intensive, and interpretation is difficult, especially in patients with mild disorders. In addition, interuser variability in performance of the assays, including the currently accepted gold standard, light transmission aggregometry, makes the results difficult to compare between laboratories. Furthermore, a similar pattern of mucocutaneous bleeding is seen in disorders in other components of the hemostatic pathway, including type 1 von Willebrand disease (VWD). We have undertaken an extensive investigation of patients with clinically diagnosed excessive bleeding, using a genotyping and platelet phenotyping approach based on lumi-aggregometry, and other specialist tests of platelet function, in combination with Sanger and next-generation sequencing (NGS). We found a functional defect in ~ 60% of patients, the majority being associated with feedback pathways of platelet activation. Function-disrupting mutations were identified in known and novel genes, and coinheritance with other genetic disorders of hemostasis, including type 1 VWD, was shown. A significant number of mutations are heterozygous and unlikely to cause extensive bleeding in isolation, consistent with incomplete penetrance of inheritance of bleeding disorders and a multifactorial etiology for excessive bleeding in many patients. Mucocutaneous bleeding is a complex trait, and this has important implications for NGS in the assessment of a PFD.

A Novel ABCA12 Mutation in Two Families with Congenital Ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) is a rare genetically heterogeneous disorder characterized by hyperkeratosis in addition to dry, scaly skin. There are six genes currently known to be associated with the disease. Exome sequencing data for two affected individuals with ichthyosis from two apparently unrelated consanguineous Pakistani families was analysed. Potential candidate mutations were analysed in additional family members to determine if the putative mutation segregated with disease status. A novel mutation (c.G4676T, p.Gly1559Val) in ABCA12 occurred at a highly conserved residue, segregated with disease status in both families, and was not detected in 143 control chromosomes. Genotyping with microsatellite markers demonstrated a partial common haplotype in the two families, and a common founder mutation could not be excluded. Comparison to previously reported cases was consistent with the hypothesis that severe loss of function ABCA12 mutations are associated with Harlequin Ichthyosis and missense mutations are preferentially associated with milder phenotypes. In addition to identifying a possible founder mutation, this paper illustrates how advances in genome sequencing technologies could be utilised to rapidly elucidate the molecular basis of inherited skin diseases which can be caused by mutations in multiple disease genes.