Rein P. Stulp

Revertant Mosaicism in Epidermolysis Bullosa Caused by Mitotic Gene Conversion

Mitotic gene conversion acting as reverse mutation has not been previously demonstrated in human. We report here that the revertant mosaicism of a compound heterozygous proband with an autosomal recessive genodermatosis, generalized atrophic benign epidermolysis bullosa, is caused by mitotic gene conversion of one of the two mutated COL17A1 alleles. Specifically, the maternal allele surrounding the mutation site on COL17A1 (1706delA) showed reversion of the mutation and loss of heterozygosity along a tract of at least 381 bp in revertant keratinocytes derived from clinically unaffected skin patches; the paternal mutation (R1226X) remained present in all cell samples. Revertant mosaicism represents a way of natural gene therapy.

RET and GDNF gene scanning in Hirschsprung patients using two dual denaturing gel systems.

Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal obstruction due to an absence of intramural ganglia along variable lengths of the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF gene, and encoding one of the RET ligands, either alone or in combination with RET mutations, can also cause HSCR, as can mutations in four other genes (EDN3, EDNRB, ECE1, and SOX10). The rare mutations in the latter four genes, however, are more or less restricted to HSCR associated with specific phenotypes. We have developed a novel comprehensive mutation detection system to analyse all but three amplicons of the RET and GDNF genes, based on denaturing gradient gel electrophoresis. We make use of two urea‐formamide gradients on top of each other, allowing mutation detection over a broad range of melting temperatures. For the three remaining (GC‐rich) PCR fragments we use a combination of DGGE and constant denaturing gel electrophoresis (CDGE). These two dual gel systems substantially facilitate mutation scanning of RET and GDNF, and may also serve as a model to develop mutation detection systems for other disease genes. In a screening of 95 HSCR patients, RET mutations were found in nine out of 17 familial cases (53%), all containing long segment HSCR. In 11 of 78 sporadic cases (14%), none had long segment HSCR. Only one GDNF mutation was found, in a sporadic case. Hum Mutat 15:418–429, 2000.

Three novel KCNA1 mutations in episodic ataxia type I families

Hereditary paroxysmal ataxia, or episodic ataxia (EA), is a rare, genetically heterogeneous neurological disorder characterized by attacks of generalized ataxia. By direct sequence analysis, a different missense mutation of the potassium channel gene (KCNA1) has been identified in three families with EA.

No mutations found by RET mutation scanning in sporadic and hereditary neuroblastoma

Neuroblastoma occasionally occurs in diseases associated with abnormal neurocrest differentiation, e.g. Hirschsprung disease. Expression studies in developing mice suggest that the proto-oncogeneRET plays a role in neurocrest differentiation. In humans expression ofRET is limited to certain tumor types, including neuroblastoma, that derive from migrating neural crest cells. Mutations ofRET are found associated with Hirschsprung disease. These data prompted us to investigate expression ofRET and to search for gene mutations in neuroblastoma. Out of 16 neuroblastoma cell lines analyzed, 9 show clear expression ofRET in a Northern blot analysis. In a single-strand conformation polymorphism (SSCP) analysis of all exons, no mutations were detected other than neutral polymorphisms. In a patient with neuroblastoma, from a family in which different neurocrestopathies, including neuroblastoma and Hirschsprung disease, had occurred, we also failed to detect RET mutations. Possibly, expression ofRET in neuroblastoma merely reflects the differentiation status of the tumor cells. The absence of mutations suggests thatRET does not play a crucial role in the tumorigenesis of neuroblastoma.

A novel pathogenic MLH1 missense mutation, c.112A > C, p.Asn38His, in six families with Lynch syndrome

BackgroundAn unclassified variant (UV) in exon 1 of the MLH1 gene, c.112A > C, p.Asn38His, was found in six families who meet diagnostic criteria for Lynch syndrome. The pathogenicity of this variant was unknown. We aim to elucidate the pathogenicity of this MLH1 variant in order to counsel these families adequately and to enable predictive testing in healthy at-risk relatives.MethodsWe studied clinical data, microsatellite instability and immunohistochemical staining of MMR proteins, and performed genealogy, haplotype analysis and DNA testing of control samples.ResultsThe UV showed co-segregation with the disease in all families. All investigated tumors showed a microsatellite instable pattern. Immunohistochemical data were variable among tested tumors. Three families had a common ancestor and all families originated from the same geographical area in The Netherlands. Haplotype analysis showed a common haplotype in all six families.ConclusionsWe conclude that the MLH1 variant is a pathogenic mutation and genealogy and haplotype analysis results strongly suggest that it is a Dutch founder mutation. Our findings imply that predictive testing can be offered to healthy family members. The immunohistochemical data of MMR protein expression show that interpreting these results in case of a missense mutation should be done with caution.

Implications of intragenic marker homozygosity and haplotype sharing in a rare autosomal recessive disorder: the example of the collagen type XVII (COL17A1) locus in generalised atrophic benign epidermolysis bullosa

Abstract Generalised atrophic benign epidermolysis bullosa (GABEB) is a form of junctional epidermolysis bullosa with a recessive mode of inheritance. The gene considered likely to be involved in this disease is COL17A1, since in the majority of GABEB patients the product of that gene, the 180-kD bullous pemphigoid antigen (BP180), is undetectable in skin. We have identified an intragenic COL17A1 microsatellite marker for which 83% of randomly selected control individuals are heterozygous. We observed homozygosity for different alleles of this marker in five out of six collagen type XVII-negative GABEB patients of different European descent. Five of the six COL17A1 alleles of three patients originating from the eastern part of The Netherlands were identical, as were the haplotypes including flanking markers. The 2342delG mutation was identified in all these five alleles. This confirms the expectation that due to genetic drift and hidden inbreeding for an autosomal recessive disorder with low gene frequency, such as collagen type XVII-negative GABEB, most disease alleles from a restricted geographical area will be “identical by descent”. Our results demonstrate that involvement of a candidate gene can be confirmed by looking for identity by descent of highly informative intragenic markers.

Absence of mutations in the RET gene in acute myeloid leukemia

Abstract Expression of the tyrosine kinase receptor RET has previously been detected in normal hematopoietic cells, and especially in cells of the myeloid lineage. Furthermore, RET was shown to be differentially expressed in acute myeloid leukemia (AML), a disease characterized by excessive cell growth and aberrant maturation of cells, with the highest levels of expression in leukemias with monocytic differentiation. RET is known to be expressed in cells from the excretory system and from the developing central and peripheral nervous system. Both activating and inactivating aberrations in the RET gene have been detected in disorders derived from these tissues. To investigate whether the differential expression is a primary defect in AML, the presence of RET alterations was scanned by Southern blot analysis on DNA of blasts obtained from 17 AML patients. However, no RET gene aberrations were found. Subsequently, denaturing gradient gel electrophoresis (DGGE) analysis was performed on the DNA of blasts from ten selected cases. All five variants detected turned out to represent neutral DNA polymorphisms, including a novel polymorphism in exon 14. Since we were unable to detect mutations of RET in AML, it is unlikely that it plays an important role in leukemogenesis.

Clinical Vignette: Early-Onset Head and Neck Cancer: Beware of Fanconi Anaemia!

Without knowing any further clinical details, the possibility of Fanconi anaemia (FA) [1] crossed our minds. Oral cancer is a very rare diagnosis in young individuals from the Dutch general population, with incidence rates per 100,000 person-years being smaller than 0.5 for cancer of the palate or tongue in the 30-34-year age group [2]. Fanconi anaemia, an autosomal recessive disorder, is associated with early-onset head and neck cancer and a range of other neoplasms [3-5]. The fact that no congenital anomalies had been mentioned in the referral letter did not exclude the possibility of FA, because either the otolaryngologist might not have realized their importance with respect to a genetic diagnosis, or they simply had not been present as would be expected in up to one-third of FA patients [6]. We were concerned about the scheduled treatment because of the strongly increased risk of complications after therapy with mutagenic agents (chemical or radiation) in FA patients [3,7-10]. Immediately but unsuccessfully, we tried to contact the referring physician by telephone. An urgent letter was therefore sent to this physician, mentioning the fact that FA was at least a theoretical possibility in this patient (who we had not seen yet) and might severely complicate his treatment. An offer to quickly test the patients lymphocytes for signs of FA (increased chromosomal breakage and structural rearrangements after culture with the DNA cross-linking agent mitomycin C) was included. We also contacted the family physician who informed us that the patient had declined further treatment and had already died around the time we received the letter of referral. Upon explaining the reason for our inquiry, we were surprised to learn that the deceased brother of the patient had indeed been diagnosed with FA as a child! The old medical files of both brothers were retrieved. The brother of the referred patient had been diagnosed with Fanconi anaemia in his late teens on the basis of pancytopenia, increased HbF, short stature (<P3, skeletal age several years behind calendar age), horse-shoe kidney, axillary and inguinal hyperpigmentation, a few cafe-au-lait spots on the skin and increased chromosomal breakage typical for FA. He was diagnosed with squamous cell cancer of the soft palate at the age of 31 and died at the age of 34 (no details on treatment are available). His younger brother (the referred patient) had been diagnosed as a teen with short stature (<P3, skeletal age several years behind calendar age), persistent mild leukopenia and thrombopenia and a severe congenital sub-pelvic stenosis of one of his kidneys. Although the patient had not been tested for chromosomal breakage, it is fair to assume that he had FA as well. For reasons unknown to us, the referring otolaryngologist had not been aware of the diagnosis of FA in this family or had not realised its association with head and neck cancer. The parents and their two healthy adult children (i.e. sibs of the deceased patients) were seen at our familial cancer clinic. At their own request, the children were tested for chromosomal breakage and rearrangements, with normal results. The parents as well as their children expressed sincere amazement after learning about the diagnosis of FA in their family and its association with head and neck cancer and other neoplasms. The parents could not recollect having ever heard about this disorder before, although we assume that the paediatricians have discussed it with them many years ago. No follow-up aimed at early detection of cancer had apparently been performed in their adult sons with FA. Current FA guidelines do advise such surveillance [5,11] and include gynaecological examination and Pap smears, annual rectal examination, and frequent dental and oropharyngeal examinations. Annual oesophageal endoscopy has also been recommended. We cannot help feeling that if medical (oncological) surveillance had been more aggressively pursued after childhood in these two brothers with Fanconi anaemia, their early deaths might have been prevented. This case also makes one wonder which proportion of FA patients that reach adulthood and are lost to paediatric follow-up, are actually under recommended oncological surveillance. We would like to conclude this paper on a more positive note. This clinical history illustrates the increasing trend we observe among clinicians from disciplines that did not traditionally refer many patients for (onco)genetic analysis, to recognise early-onset cancer as an important warning sign of inherited cancer predisposition and to consult a family cancer clinic in those cases. An overview of referral guidelines for cancer genetics consultations has recently been published [12]. To those we would like to add that in the case of early-onset head and neck cancer, the possibility of Fanconi anaemia, and therefore referral, should always be very seriously considered.