Karen E. Heath

Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions (?Döhle-like? bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8?10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.The autosomal dominant, giant-platelet disorders1, May-Hegglin anomaly2,3 (MHA; MIM 155100), Fechtner syndrome4 (FTNS; MIM 153640) and Sebastian syndrome5 (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions (?Döhle-like? bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis4. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 810), which is expressed in platelets9 and upregulated during granulocyte differentiation10. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

Low-density lipoprotein receptor gene (LDLR) world-wide website in familial hypercholesterolaemia: update, new features and mutation analysis

Mutations in the low density lipoprotein receptor gene (LDLR) cause familial hypercholesterolaemia (FH). The FH website (http://www.ucl. ac.uk/fh) has been updated to provide various functions enabling the analysis of the large number of LDLR mutations. To date, 683 LDLR mutations have been reported; of these 58.9% are missense mutations, 21.1% minor rearrangements, 13.5% major rearrangements and 6.6% splice site mutations. Of the 402 missense mutations, only 11.4% occurred at CpG sites. The majority of mutations were found in two functional domains, the ligand binding domain (42%) and the epidermal growth factor (EGF) precursor-like domain (47%). This report describes new features of the FH website and assesses the spectrum of mutations reported to date.

A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom

A genetic diagnostic service for familial hypercholesterolaemia (FH) has been established over the last 4 years in the Clinical Molecular Genetics Laboratory at Great Ormond Street Hospital for Children NHS Trust (GOSH), London. In total there have been 368 referrals; 227 probands and 141 family members, which have come from a number of lipid clinics and from general practitioners. FH is caused by mutations in the low-density lipoprotein receptor gene (LDLR) and these are analysed by SSCP, DNA sequencing and direct assays. The clinically indistinguishable disorder, familial defective apolipoprotein B100 (FDB) is caused by one of three mutations in the apolipoprotein B100 gene (APOB) which are analysed by direct assays. Mutations predicted to be pathogenic were found in 76 probands, 67 in LDLR (23 previously undescribed) and nine in APOB. The mutation detection rate was 53% in paediatric probands, 32% in adults with a ‘definite’ FH diagnosis (tendon xanthoma positive) and 14% in adults with a ‘possible’ FH diagnosis (tendon xanthoma negative). The predicted loss of sensitivity that would result from reducing the number of exons tested has been assessed, and a molecular screening strategy suitable for UK patients is proposed. A similar strategy may be useful for other countries where genetic heterogeneity results in a wide mutation spectrum for FH.

Universal primer quantitative fluorescent multiplex (UPQFM) PCR: a method to detect major and minor rearrangements of the low density lipoprotein receptor gene

A method based on quantitative fluorescent multiplex PCR has been developed to detect major rearrangements of the low density lipoprotein receptor gene (LDLR) which account for ∼5% of mutations. The method involves two PCR reactions; the first (P1) amplifies the selected exons using unique primer sequences tagged with newly designed universal primers, while the second (P2) amplifies the P1 amplicons using the universal primers. One of the P2 universal primers is labelled with a fluorescent dye which is incorporated into the PCR products which are then electrophoresed on an ABI DNA sequencer. The relative amounts of the amplified peak areas are determined and compared to ratios obtained for DNA from four normal controls and known major rearrangements. The multiplex set developed is based on LDLR exons 3, 5, 8, 14, and 17 and 86% of reported major rearrangements would be detectable by this assay as well as any deletions and insertions of greater than 1 bp. The method was evaluated using DNA from 15 reported deletions and duplications which were all correctly identified. Two groups of UK patients with a clinical diagnosis of familial hypercholesterolaemia (FH) and where no mutation had been identified inLDLR or APOB (14 children and 42 adults) were screened for the presence of majorLDLR rearrangements by this assay. Three major rearrangements were detected and a 4 bp duplication was identified in a fourth patient. Since it avoids the problems associated with Southern blotting, this method will be useful for detecting gene rearrangements.

CDKN1C (p57Kip2) analysis in Beckwith–Wiedemann syndrome (BWS) patients: Genotype–phenotype correlations, novel mutations, and polymorphisms

Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by macroglossia, macrosomia, and abdominal wall defects. It is a multigenic disorder caused in most patients by alterations in growth regulatory genes. A small number of individuals with BWS (5–10%) have mutations in CDKN1C, a cyclin‐dependent kinase inhibitor of G1 cyclin complexes that functions as a negative regulator of cellular growth and proliferation. Here, we report on eight patients with BWS and CDKN1C mutations and review previous reported cases. We analyzed 72 patients (50 BWS, 17 with isolated hemihyperplasia (IH), three with omphalocele, and two with macroglossia) for CDKN1C defects with the aim to search for new mutations and to define genotype–phenotype correlations. Our findings suggest that BWS patients with CDKN1C mutations have a different pattern of clinical malformations than those with other molecular defects. Polydactyly, genital abnormalities, extra nipple, and cleft palate are more frequently observed in BWS with mutations in CDKN1C. The clinical observation of these malformations may help to decide which genetic characterization should be undertaken (i.e., CDKN1C screening), thus optimizing the laboratory evaluation for BWS.

A World Wide Web Site for Low-Density Lipoprotein Receptor Gene Mutations in Familial Hypercholesterolemia: Sequence-Based, Tabular, and Direct Submission Data Handling☆

Familial hypercholesterolemia is an autosomal dominant inherited condition characterized by a mutation in the low-density lipoprotein receptor (LDLR) gene. A database has been set up on the World Wide Web for mutations in the LDLR gene.

Identification of the first recurrent PAR1 deletion in Léri-Weill dyschondrosteosis and idiopathic short stature reveals the presence of a novel SHOX enhancer

Background SHOX, located in the pseudoautosomal region 1 (PAR1) of the sexual chromosomes, encodes a transcription factor implicated in human growth. Defects in SHOX or its enhancers have been observed in ∼60% of Leri-Weill dyschondrosteosis (LWD) patients, a skeletal dysplasia characterised by short stature and/or the characteristic Madelung deformity, and in 2–5% of idiopathic short stature (ISS). To identify the molecular defect in the remaining genetically undiagnosed LWD and ISS patients, this study screened previously unanalysed PAR1 regions in 124 LWD and 576 ISS probands. Methods PAR1 screening was undertaken by multiplex ligation dependent probe amplification (MLPA). Copy number alterations were subsequently confirmed and delimited by locus-specific custom-designed MLPA, array comparative genomic hybridisation (CGH) and breakpoint junction PCR/sequencing. Results A recurrent PAR1 deletion downstream of SHOX spanning 47543 bp with identical breakpoints was identified in 19 LWD (15.3%) and 11 ISS (1.9%) probands, from 30 unrelated families. Eight evolutionarily conserved regions (ECRs 1–8) identified within the deleted sequence were evaluated for SHOX regulatory activity by means of chromosome conformation capture (3C) in chicken embryo limbs and luciferase reporter assays in human U2OS osteosarcoma cells. The 3C assay indicated potential SHOX regulatory activity by ECR1, which was subsequently confirmed to act as a SHOX enhancer, operating in an orientation and position independent manner, in human U2OS cells. Conclusions This study has identified the first recurrent PAR1 deletion in LWD and ISS, which results in the loss of a previously uncharacterised SHOX enhancer. The loss of this enhancer may decrease SHOX transcription, resulting in LWD or ISS due to SHOX haploinsufficiency.

Primary Acid-Labile Subunit Deficiency due to Recessive IGFALS Mutations Results in Postnatal Growth Deficit Associated with Low Circulating Insulin Growth Factor (IGF)-I, IGF Binding Protein-3 Levels, and Hyperinsulinemia

CONTEXT Up to 90% of circulating IGF-I and IGF-II are carried bound to either IGF binding protein (IGFBP)-3 or IGFBP-5 and the acid-labile subunit (ALS) in the form of tertiary complexes that extend their circulating half-life. Three cases of complete ALS deficiency have been recently reported in short-stature patients with very low circulating IGF-I and IGFBP-3 levels who presented with homozygous or compound heterozygous mutations in the ALS encoding gene (IGFALS; 16p13.3), thus supporting a role for ALS in the regulation of the bioavailability of IGFs during postnatal growth. OBJECTIVE We present the molecular and clinical characterization of two novel IGFALS mutations that caused complete ALS deficiency in three unrelated patients with postnatal growth deficit, low IGF-I and IGFBP-3 levels, and no GH deficiency. RESULTS IGFALS mutation screening identified a novel homozygous IGFALS missense mutation, which altered a conserved residue, N276S, in two of the probands. The third proband presented a novel homozygous nonsense mutation, Q320X, that is predicted to generate a severely truncated ALS protein. The affected probands presented a similar phenotype characterized by a moderate postnatal growth deficit associated with undetectable ALS, low IGF-I, IGF-II, and IGFBP-3, and hyperinsulinemia, and, in two cases, delayed puberty. CONCLUSIONS Primary ALS deficiency due to IGFALS mutations should be considered as a possible cause of postnatal growth deficit in IGF-I-deficient patients in the absence of GH deficiency or insensitivity. Determination of serum ALS levels and basal insulinemia can be helpful in the differential diagnosis of patients with idiopathic IGF-I deficiency.

Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature

Short stature homeobox-containing gene, MIM 312865 (SHOX) is located within the pseudoautosomal region 1 (PAR1) of the sex chromosomes. Mutations in SHOX or its downstream transcriptional regulatory elements represent the underlying molecular defect in ∼60% of Léri-Weill dyschondrosteosis (LWD) and ∼5–15% of idiopathic short stature (ISS) patients. Recently, three novel enhancer elements have been identified upstream of SHOX but to date, no PAR1 deletions upstream of SHOX have been observed that only encompass these enhancers in LWD or ISS patients. We set out to search for genetic alterations of the upstream SHOX regulatory elements in 63 LWD and 100 ISS patients with no known alteration in SHOX or the downstream enhancer regions using a specifically designed MLPA assay, which covers the PAR1 upstream of SHOX. An upstream SHOX deletion was identified in an ISS proband and her affected father. The deletion was confirmed and delimited by array-CGH, to extend ∼286 kb. The deletion included two of the upstream SHOX enhancers without affecting SHOX. The 13.3-year-old proband had proportionate short stature with normal GH and IGF-I levels. In conclusion, we have identified the first PAR1 deletion encompassing only the upstream SHOX transcription regulatory elements in a family with ISS. The loss of these elements may result in SHOX haploinsufficiency because of decreased SHOX transcription. Therefore, this upstream region should be included in the routine analysis of PAR1 in patients with LWD, LMD and ISS.

The Gene for May-Hegglin Anomaly Localizes to a <1-Mb Region on Chromosome 22q12.3-13.1

The May-Hegglin anomaly (MHA) is an autosomal dominant platelet disorder of unknown etiology. It is characterized by thrombocytopenia, giant platelets, and leukocyte inclusion bodies, and affected heterozygotes are predisposed to bleeding episodes. The MHA gene has recently been localized, by means of linkage analysis, to a 13.6-cM region on chromosome 22, and the complete chromosome 22 sequence has been reported. We recently performed a genome scan for the MHA gene in 29 members of a large, multigenerational Italian family, and we now confirm that the MHA locus is on chromosome 22q12. 3-13.1. The maximal two-point LOD score of 4.50 was achieved with the use of marker D22S283, at a recombination fraction of.05. Haplotype analysis narrowed the MHA critical region to 6.6 cM between markers D22S683 and D22S1177. It is of note that the chromosome 22 sequence allowed all markers to be ordered correctly, identified all the candidate genes and predicted genes, and specifically determined the physical size of the MHA region to be 0. 7 Mb. These results significantly narrow the region in which the MHA gene is located, and they represent the first use of chromosome 22 data to positionally clone a disease gene.