Genome sequencing reveals underdiagnosis of primary ciliary dyskinesia in bronchiectasis.

BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.

Partial cDNA sequence for the donkey chorionic gonadotrophin-beta subunit suggests evolution from an ancestral LH-beta gene.

A 246 bp cDNA clone representing the C-terminal region of the donkey (Equus asinus) chorionic gonadotrophin (CG)-beta subunit was isolated from a placental library. The transcript contained the 3' untranslated region and 42% of the CG-beta subunit coding region (amino acid residues 85-146 of the mature peptide). Comparison of the deduced donkey amino acid sequence with the published horse CG-beta subunit protein sequence (where they overlapped) revealed an overall homology of 61%. However, most of the differences were in the C-terminal extension, which is thought not to be important for gonadotrophic activity, and appeared to be due to two nucleotide insertions in the donkey sequence (compared with a deduced horse nucleotide sequence) leading to a reading-frame shift. Amino acid homology in the disulphide 'core' region was 81%. Some of the differences in this region were in the 'determinant loop' (residues 93-100) and these are interpreted in relation to the observed biological activities of horse and donkey CG. The deduced amino acid sequence of the donkey cDNA indicated that it was larger than the majority of gonadotrophin-beta subunits due to a C-terminal extension. Primate and horse CG (and horse LH) beta subunits have analogous C-terminal extensions. The extension in the donkey subunit is 25 amino acid residues in length, compared with 28 in the horse and 24 in man. Comparisons with other available subunit DNA sequences indicated that, like the human CG-beta gene, the donkey gene probably evolved from an ancestral LH-like beta gene, following nucleotide deletions that allowed readthrough into previously untranslated DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide (cDNA) sequence encoding the horse gonadotrophin alpha-subunit.

Several cDNA clones corresponding to mRNA for the alpha-subunit of the horse (Equus caballus) pituitary and placental (chorionic) gonadotrophic hormones have been isolated and sequenced. Polyadenylated mRNA was purified from horse pituitary glands (the source of FSH and LH) and horse placental tissues (the source of chorionic gonadotrophin; CG). The mRNA preparations were characterized by in-vitro translation and Northern hybridization techniques using human and ovine gonadotrophin cDNA clones as probes. Complementary DNA libraries were created from the pituitary and placental mRNAs and a human CG alpha-subunit probe was used to isolate several horse alpha-subunit cDNA clones. The alpha-subunit nucleotide sequence from both sources of tissue was identical, thereby indicating that in the horse (as in man) the same gonadotrophin alpha-subunit gene is expressed in the pituitary and placenta. Our results are consistent with transcription of a single alpha-subunit gene for all the glycoprotein hormones in the horse, and we suggest that the reported differences between the horse CG and FSH alpha-subunit amino acid sequences determined by conventional peptide sequencing methods arose due to errors in the FSH alpha-subunit sequence. Comparison of the deduced amino acid sequence of the horse alpha-subunit with that of other alpha-subunit sequences indicated a number of significant differences which may be related to the unusual receptor-binding properties of the equine gonadotrophins.

Update and analysis of the University College London low density lipoprotein receptor familial hypercholesterolemia database.

Familial hypercholesterolemia (FH) (OMIM 143890) is most commonly caused by variations in the LDLR gene which encodes the receptor for Low Density Lipoprotein (LDL) cholesterol particles. We have updated the University College London (UCL) LDLR FH database (http://www.ucl.ac.uk/ldlr) by adding variants reported in the literature since 2001, converting existing entries to standard nomenclature, and transferring the database to the Leiden Open Source Variation Database (LOVD) platform. As of July 2007 the database listed 1066 unique LDLR gene events. Sixty five percent (n = 689) of the variants are DNA substitutions, 24% (n = 260) small DNA rearrangements (<100bp) and 11% (n = 117) large DNA rearrangements (>100bp), proportions which are similar to those reported in the 2001 database (n = 683, 62%, 24% and 14% respectively). The DNA substitutions and small rearrangements occur along the length of the gene, with 24 in the promoter region, 86 in intronic sequences and 839 in the exons (93 nonsense variants, 499 missense variants and 247 small rearrangements). These occur in all exons, with the highest proportion (20%) in exon 4 (186/949); this exon is the largest and codes for the critical ligand binding region, where any missense variant is likely to be pathogenic. Using the PolyPhen and SIFT prediction computer programmes 87% of the missense variants are predicted to have a deleterious effect on LDLR activity, and it is probable that at least 48% of the remainder are also pathogenic, but their role in FH causation requires confirmation by in vitro or family studies.

Use of fluorescent in situ hybridisation to confirm trisomy of chromosome region 1q32-qter as the sole karyotypic defect in a colon cancer cell line.

The sole chromosome defect in a colon cancer cell line derived from a patient with inherited nonpolyposis colorectal cancer was karyotypically designated as 46,XY,-13,+der(13)t(1;13)(q32.1;p11) on the basis of banding homology. We have obtained molecular confirmation that the additional chromosome material is derived from chromosome region 1q32-qter by the use of a highly specific fluorescent in situ hybridisation technique on G-banded chromosomes and also by Southern hybridisation.

Application of recombinant DNA techniques to structure-function studies of equine protein hormones.

Complementary (c)DNA libraries have been made from horse pituitary gland and endometrial cup tissues with the aim of isolating the genes for the horse gonadotrophins (FSH, LH and CG) and growth hormone (GH). Southern (DNA) and Northern (RNA) blotting techniques were used to demonstrate that several heterologous (human and ovine) cDNA probes would be adequate for isolating the horse genes. A human cDNA probe was then used to isolate the horse gonadotrophin alpha-subunit cDNA from the pituitary and endometrial cup libraries. The nucleotide sequences from both tissue sources were identical, thereby confirming that there is a single gene for the alpha-subunit in the horse. As soon as the gonadotrophin beta-subunit sequences become available, they will be expressed along with the alpha-subunit to obtain biologically active hormones. Horse GH cDNA has also been isolated by using a sheep cDNA probe and its DNA sequence has been determined. It was subsequently used to demonstrate that horse endometrial cups, but not the allantochorion taken at the same stage of gestation, contains mRNA for a GH-like protein. This mRNA could be due to a low level of expression of the GH gene or to expression of the gene(s) for a GH-related protein such as prolactin or placental lactogen.

Chromosome 5 allele loss in familial and sporadic colorectal adenomas.

DNA extracted from familial and sporadic colorectal neoplasms was compared with constitutional DNA using a range of hypervariable locus specific probes to assess the extent of allele loss during conversion to malignancy. Chromosome 5 allele loss was observed in 23% of carcinoma samples, as previously found by others. However, we have been able to show for the first time loss of the D5S43 locus on chromosome 5 in adenomas from three patients, two of whom had the precancerous condition adenomatous polyposis coli (APC). These results suggest significant genetic changes involving chromosome 5 are occurring in benign adenomas. Probes for chromosome 1 (loci D1S7 and D1S8) and for chromosome 7 (loci D7S21 and D7S22) revealed no notable alterations in the adenoma samples. Complete loss of alleles for loci on chromosome 7 was not observed in carcinomas but reduced intensity of one parental allele was found in three specimens one of which was known to have multiple copies of this chromosome. Results using probes for chromosome 1 suggest that deletion of the D1S7 or D1S8 loci is not a common event in colorectal carcinogenesis. Loss of chromosome 5 alleles in adenomas from APC patients provides evidence in support of Knudson's hypothesis.

Molecular genetic evidence for the delineation of a more severe form of familial adenomatous polyposis which results from fresh mutation.

Familial adenomatous polyposis, an inherited pre-malignant condition, is caused by mutation in the adenomatous polyposis coli (APC) gene at chromosome 5q22. The lifetime risk of carcinoma approaches 100%, with an average age at death from cancer of 40 years, allowing most patients to complete reproduction. Since there is no evidence for a rising incidence, this is at variance with an apparently high mutation rate. We present evidence for the delineation of a severe form, which hitherto has largely been maintained by fresh mutation. An atypically high frequency of loss of heterozygosity at chromosome 5q22 in small adenomas correlated with an early age of onset or malignancy in two patients, both due to fresh mutation. In both cases, the mutation in APC was shown to be a commonly occurring deletion, leading us to postulate the co-existence of a modifying gene.

Exclusion of ZFM1 as a candidate gene for multiple endocrine neoplasia type 1 (MEN1).

The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping and linkage studies and a 3.8-cM region flanked by PYGM and D11S97 has been defined. The zinc finger in the MEN1 locus (ZFM1) gene, which has also been mapped to this region, represents a candidate gene for MEN1. The ZFM1 gene, which consists of 14 exons, encodes a 623-amino acid protein and exons 2, 8 and 12 encode the putative nuclear localisation signal, a zinc finger motif, and a proline-rich region, respectively. We have investigated these potentially functional regions for germ-line mutations by single-stranded conformational polymorphism (SSCP) analysis in 64 unrelated MEN1 patients. In addition, we performed DNA sequence analysis of all the 14 exons and 13 of the 26 exon-intron boundaries in four unrelated MEN1 patients. A 6-bp deletion that resulted in the loss of two proline residues at codons 479 and 480 in exon 12 was found in one MEN1 patient. However, this did not co-segregate with MEN1 in the family and represented a rare polymorphism. Analysis by SSCP, DNA sequencing, northern blotting, Southern blotting and pulsed field gel electrophoresis revealed no additional genetic abnormalities of ZFM1 in the other MEN1 patients. Thus, our results indicate that ZFM1 is excluded as a candidate gene for MEN1.

Construction of a YAC contig and an STS map spanning 3.6 megabase pairs in Xp22.1.

We have constructed a 3.6 Mb sequence tagged sites (STS)-based yeast artificial chromosome (YAC) contig, consisting of 58 individual YAC clones, spanning the region PDHA1 and DXS451 on Xp22.1. In addition to establishing the order of PDHA1, ISPK-1, DXS2504, DXS1528 and the 13 known polymorphic loci as Xpter-PDHA1-DXS443-DXS3424-ISPK-1-DXS12 29-DXS2504-DXS1528-DXS365-DXS7101- DXS1683-DXS1052-DXS274-DXS92-DXS1226-DX S41-DXS989-DXS451-Xcen, we have also developed 35 novel STSs from YAC end clones. These results provide a high density of STS markers (approximately 1 per 70 kb). Furthermore, a detailed long-range restriction map of the contig has been constructed with rare-cutter enzymes and this has refined and verified the physical distances between markers inferred from YAC sizes and their STS content. The integration of the physical mapping data with previous genetic mapping data and the use of STSs and non-chimeric YAC clones reported here should facilitate the construction of a transcript map of this region and the positional cloning of disease genes in this portion of Xp22.1.

Genetic mapping studies of 40 loci and 23 cosmids in chromosome 11p13-11q13, and exclusion of mu-calpain as the multiple endocrine neoplasia type 1 gene.

Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11q13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MEN1-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871-(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM- SEA-D11S913-D11S970-D11S97- D11S146-INT2-D11S971-D11S533-11qter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding mu-calpain (CAPN1) located CAPN1 to 11q13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11q13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region.