Chcm Buys

Oncological implications of RET gene mutations in Hirschsprung’s disease

Background—Germline mutations of the RET proto-oncogene identical to those found in the tumour predisposition syndrome multiple endocrine neoplasia type 2A (MEN2A), were detected in 2.5–5% of sporadic and familial cases of Hirschsprung’s disease. Some patients with Hirschsprung’s disease may therefore be exposed to a highly increased risk of tumours. Aims—To define clinical use of RET gene testing in Hirschsprung’s disease and related patient management from an oncological point of view. Methods—Sixty patients with Hirschsprung’s disease were screened for RET mutations. In three, MEN2A type RET mutations were detected. Case reports for these three patients are presented. Results and conclusions—Only 22 families or sporadic patients with Hirschsprung’s disease and MEN2A type RET mutations have been reported. Therefore, it is difficult to predict tumour risk for patients with familial or sporadic Hirschsprung’s disease, and their relatives, who carry these mutations. For these mutation carriers, periodic screening for tumours as in MEN2A is advised, but prophylactic thyroidectomy is offered hesitantly. RET gene testing in familial or sporadic Hirschsprung’s disease is not recommended at present outside a complete clinical research setting. In combined MEN2A/Hirschsprung’s disease families RET gene testing, tumour screening, and prophylactic thyroidectomy are indicated as in MEN2A.

Glycogen storage disease type Ia: recent experience with mutation analysis, a summary of mutations reported in the literature and a newly developed diagnostic flowchart

Abstract We studied the glucose-6-phosphatase (G6Pase) gene of 30 unrelated glycogen storage disease type Ia (GSD Ia) patients using single strand conformational polymorphism (SSCP) prior to automated sequencing of exons revealing an aberrant SSCP pattern. In all patients we could identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure. A total of 14 different mutations were identified. R83C (16/60), 158delC (12/60), Q347X (7/60), R170X (6/60) and ΔF327 (4/60) were found most frequently. Nine other mutations accounted for the other 15 mutant alleles. Two DNA-based prenatal diagnoses were performed successfully. At present, 56 mutations in the G6Pase gene have been reported in 300 unrelated GSD Ia patients and an overview of these mutations is presented. Evidence for a clear genotype-phenotype correlation could be established neither from our data nor from those in the literature. With increased knowledge about the genetic basis of GSD Ia and GSD Ib and the high detection rate of mutations, it is our opinion that the diagnoses GSD Ia and GSD Ib can usually be based on clinical and biochemical abnormalities combined with mutation analysis instead of enzyme assays in liver tissue obtained by biopsy. A newly developed flowchart for the diagnosis of GSD I is presented. Conclusion Increased knowledge of the genetic basis of glycogen storage disease type I provides a DNA-based diagnosis, prenatal DNA-based diagnosis in chorionic villus samples and carrier detection.

Validation of the determination of Delta F508 mutations of the cystic fibrosis gene in over 11000 mouthwashes

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11413 blood donors and tested the mouthwashes for the ΔF508 mutation, which has an estimated frequency of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the ΔF508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 ΔF508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirm that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.

Partial 3q duplication syndrome and assignment of D3S5 to 3q25-3q28

SummaryWe report a girl with a de novo duplication of the distal part of the long arm of chromosome 3 and review the literature. Our patient had the facial characteristics and many other anomalies of the partial 3q duplication syndrome. As a hitherto undescribed symptom in partial 3q trisomy syndrome, she had microphthalmia. The karyotype of this girl was interpreted as an inverse duplication of the terminal portion of chromosome 3: 46,XX,inv dup (3)(pter-q28::q28–q25::q28-qter). Quantitative hybridisation studies with 3p and 3q probes gave a consistent 3∶2 ratio of the relative intensities of the q bands in relation to the p bands between patient and control. This confirmed the presence of a 3q duplication and delineated the location of D3S5 to 3q25–3q28.

RAPID UPTAKE BY LIVER SINUSOIDAL CELLS OF SERUM-ALBUMIN MODIFIED WITH RETENTION OF ITS COMPACT CONFORMATION

The clearance from the blood and the conformation of serum albumin modified by nitroguanidination and labeled with 125-I have been studied. Like formaldehyde-denatured albumin, but in contrast to native albumin, the nitroguanidinated derivative is rapidly cleared from the blood and taken up in lysosomes of liver sinusoidal cells. Although 94% of the free amino groups were blocked by nitroguanidination, we could not detect significant conformational changes using gel filtration, determination of reducible disulfide groups, and titration of tyrosine residues. It is concluded that extensive denaturation is no prerequisite for the uptake of albumin derivatives in liver sinusoidal cells. It is suggested that the nitroguanidinated protein, in contrast to native albumin, is bound on membrane receptors of sinusoidal cells. The nitroguanidino groups themselves might be bound on these receptors, but it seems equally possible that the blocking of positive charges of the albumin molecule or minor, local conformational changes of the protein are sufficient for the binding on the receptors.

A PROVISIONAL TRANSCRIPT MAP OF THE SPINAL MUSCULAR-ATROPHY (SMA) CRITICAL REGION

YACs from the region containing the spinal muscular atrophy (SMA) locus at 5q12 have been used as probes in a direct screening of cDNA libraries to isolate 8 cDNAs, mapped to different YAC fragments. Three clones showed complete identity to the genes for cyclin B1 (CCNB1), the p44 subunit of the transcription factor BTF2 (BTF2p44), and cofilin (CFL). Two clones showed partial identity to the β-glucuronidase gene (GLCB) and a rat integral membrane glycoprotein gene (RNINMEGLA). CFL turned out to have been identified by a pseudogene sequence. Related sequences occurred on other chromosomes. CCNB1 and BTF2p44 were given an exact location. The GLCB-like gene and the RNINMEGLA-like gene detected loci on both 5q and 5p. The remaining three cDNA clones were localized to the SMA region only. Their sequences did not show identity to any gene for which a function is already known. Two of them have now turned out to be identical to recently reported candidate genes for SMA.

Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2–3 Mb in the region q12–13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.

Assignment of the gene coding for human cytochrome c oxidase subunit VIb to chromosome 19, band q13.1, by fluorescence in situ hybridisation.

SummaryA cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence. Subsequent R-banding indicated that the cytochrome c oxidase subunit VIb gene is localized in band 19q13.1, extending the evidence that the human nuclear genes of cytochrome c oxidase are not clustered.

A Hirschsprung disease locus at 22q11

We report a boy with truncus arteriosus, dysmorphic features, developmental delay, passing hypotonia, short segment Hirschsprung disease (HSCR), and paroxysmal hypoventilation. FISH analysis showed an interstitial deletion in chromosome band 22q11.2 coinciding with the deletions found in DiGeorge syndrome and velocardiofacial syndrome. Mutation scanning of RET, GDNF, EDNRB, and EDN3, genes associated with Hirschsprung disease, showed no aberrations. Since we know of two more patients with velocardiofacial syndrome and HSCR, we hypothesise that a gene responsible for proper development of the enteric nervous system may be included in the 22q11.2 region.

Refinement by linkage analysis in two large families of the candidate region of the third locus (SCA3) for autosomal dominant cerebellar ataxia type I

The autosomal dominant cerebellar ataxias (ADCA) are clinically and genetically heterogeneous. To date, several loci (SCAI-V) have been identified for ADCA type I. We have studied two large families from the northern part of The Netherlands with ADCA type I with a broad intra-familial variation of symptoms. In both families significant linkage is shown of the disease to the markers of the SCA3 locus on chromosome 14. Through recombinations, the candidate region for SCA3 could be refined to a 13-cM range between D14S256 and D14S81. No recombinations were detected with the markers D14S291 and D14S280, which suggests that the SCA3 gene lies close to these loci. This finding will benefit the individuals at risk in these two families who are seeking predictive testing or prenatal diagnosis.