Influence of the type of F8 gene mutation on inhibitor development in a single centre cohort of severe haemophilia A patients.

The development of neutralizing antibodies against factor VIII (FVIII) is a major complication of treatment with FVIII in patients with severe haemophilia A. This study was designed to describe the relationship between the type and location of the factor 8 (F8) gene mutation and the development of clinically relevant inhibitors in patients with severe haemophilia A. We conducted a single centre cohort study among 318 consecutive patients (baseline FVIII activity level <0.01 IU mL(-1)) born between 1934 and 2007 who were treated with FVIII on at least 50 exposure days. The primary outcome was clinically relevant inhibitor development, defined as the occurrence of at least two positive inhibitor titres and a decreased recovery. Clinically relevant inhibitors were diagnosed in 14% (43) of patients (30 high-titre). The cumulative incidence of inhibitor development was 18% (35 of 200) in high-risk gene defects (67% in patients with large deletions, 30% in patients with nonsense mutations, 15% in patients with intron 1 or 22 inversions) and 7% (8 of 118) in low-risk gene defects (7% in patients with small deletions and insertions, 6% in patients with missense mutations, 8% in patients with splice site mutations). In patients with point mutations, the cumulative risk of developing inhibitors was highest in patients with mutations in the A3 and C2 domains (13% and 17% respectively). In conclusion, in agreement with earlier observations, the type and location of the F8 gene mutation were important determinants of inhibitor development in patients with severe haemophilia A.

Frequency of Von Hippel-Lindau germline mutations in classic and non-classic Von Hippel-Lindau disease identified by DNA sequencing, Southern blot analysis and multiplex ligation-dependent probe amplification.

The current clinical diagnosis of Von Hippel-Lindau (VHL) disease demands at least one specific [corrected] VHL manifestation in a patient with familial VHL disease, or, in a [corrected] sporadic patient, at least two or more hemangioblastomas or a single hemangioblastoma in combination with a typical visceral lesion. To evaluate this definition, we studied the frequency of germline VHL mutation in three patients groups: (i) multi-organ involvement (classic VHL), (ii) limited VHL manifestations meeting criteria (non-classic VHL) and (iii) patients with VHL-associated tumors not meeting current diagnostic VHL criteria. In addition, we validated multiplex ligation-dependent probe amplification (MLPA) as a rapid and reliable quantitative method for the identification of germline VHL deletions. The frequency of germline VHL mutations was very high in classic VHL cases with multi-organ involvement (95%), lower in non-classic cases that meet current diagnostic criteria but have limited VHL manifestations or single-organ involvement (24%) and low (3.3%), but tangible in cases not meeting current diagnostic VHL criteria. The detection of germline VHL mutations in patients or families with limited VHL manifestations, or single-organ involvement is relevant for follow-up of probands and early identification of at-risk relatives.

Hereditary hemorrhagic telangiectasia: ENG and ALK-1 mutations in Dutch patients.

Hereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber disease is an autosomal dominant disorder characterized by an aberrant vascular development. The resulting vascular lesions range from smaller mucocutaneous telangiectases to large visceral arteriovenous malformations, especially in the skin, lung, gastrointestinal tract and the brain. Mutations in the genes encoding endoglin (ENG, chromosome 9q34) and activin A receptor type-like kinase 1 (ALK-1, also named ACVRL1, chromosome 12q13) are associated with HHT1 and HHT2, respectively. We report here on the genetic and molecular heterogeneity found in the HHT population in the Netherlands. Probands of 104 apparently unrelated families were studied and we performed sequence analysis on both the ENG gene and ALK-1 gene. In most of the probands, we found a mutation in one of the two genes: 53% in the ENG gene and 40% in the ALK-1 gene. In 7% of the families no ENG or ALK1 mutation was found. The mutations detected were deletions, insertions, nonsense, missense and splice site mutations. The majority were novel mutations.

Short stature as the only presenting feature in a patient with an isodicentric (Y)(q11.23) and gonadoblastoma. A clinical and molecular cytogenetic study.

A 13-year-old phenotypically female patient presented with short stature (height SDS -2.6), but without any Turner stigmata or other dysmorphic features. Chromosome analysis showed mosaicism for an isodicentric (idic) (Y)(q11.23) containing cell line and a 45,X cell line. Subsequent gonadectomy revealed a left streak ovary and a right ovary of abnormal appearance, which on histological examination appeared to contain a gonadoblastoma. DNA analysis showed that the proposed critical region of the gonadoblastoma locus on the Y chromosome was contained within the patient's idic (Y). Conclusion. The case described here shows that patients with 45,X/46,X, isodicentric (Yp) mosaicism and a female phenotype (1) can lack external virilisation but still have a gonadoblastoma and (2) do not necessarily have Turner stigmata but can present with only short stature. This case also underlines the importance of karyotyping patients with unexplained short stature to enable gonadectomy if Y-derived material is detected.

Cryptic von Hippel-Lindau disease: germline mutations in patients with haemangioblastoma only.

OBJECTIVES: Central nervous system haemangioblastoma (HAB) is a major feature of von Hippel-Lindau (VHL) disease, and it is estimated that about 30% of HAB patients have VHL disease. Consequently, it is widely recommended that sporadic HAB patients are screened for clinical and radiological features of VHL disease because of the risk of multiple tumours. We investigated the frequency of VHL germline mutations in patients with HAB only with no clinical or radiological evidence of VHL disease to define the role of molecular genetic analysis in the management of such patients. METHODS: Eighty four patients with a single HAB (23 Dutch, 61 UK) and four with multiple HAB (two Dutch, two UK) were studied by direct sequencing of the coding region and quantitative Southern blotting. RESULTS: A VHL germline mutation was found in three of 69 (4.3%) single HAB patients aged 50 years or less (three of 84 (3.6%) total single HAB patients). A germline VHL mutation was detected in a 44 year old woman with a solitary cerebellar HAB, as well as in four clinically unaffected close relatives, and in two single HAB cases presenting at the ages of 29 and 36 years. Germline VHL mutations were detected in two of four cases with multiple HAB. CONCLUSIONS: Early detection of VHL disease is important to reduce morbidity and mortality and therefore we recommend that, in addition to conventional clinical and radiological investigations, VHL gene mutation analysis should be offered to all HAB patients younger than 50 years. HAB patients aged >50 years will have a lower a priori risk of VHL disease and further data are required to evaluate the role of routine molecular genetic investigations in late onset HAB cases. The failure to detect germline VHL mutations in some patients with multiple HAB may indicate the presence of somatic mosaicism or additional HAB susceptibility genes.

Management of renal cell carcinoma in von Hippel-Lindau disease.

BACKGROUND: An evaluation of nephron-sparing surgery (NSS) or radical nephrectomy (RN) for treating renal cell carcinoma (RCC) in patients with von Hippel-Lindau disease (VHL) was carried out. METHODS: Between 1976 and 1997, 10 patients with RCC from four VHL families, of whom seven were from one family, were studied by clinical and histopathological examination. Before 1991, three patients were treated using RN, and thereafter five patients were treated using NSS. Two patients were not operated on. RESULTS: RCCs in our patients showed a slow growth rate (on average 0.3 cm year-1), and asymptomatic patients presented with tumours of low-grade malignancy. In all patients, tumours were surrounded by a fibrous pseudocapsule. In 5 out of 17 tumours, pseudocapsular invasion was observed, and three of these five tumours broke through the pseudocapsule. To date, these patients have not shown a less favourable outcome than those without pseudocapsular involvement by tumour growth. Multicentricity of RCC was relatively low (4.6 lesions per kidney). In two of the three RN patients, only a single satellite lesion, in the direct vicinity of a RCC, was found in one kidney. Six tumours (1.8-5.5 cm) were enucleated by NSS. During a mean follow-up of 30 months, renal function in these patients was well preserved. CONCLUSIONS: In our patients, RCCs grew slowly, were of low grade, had a dense fibrous pseudocapsule and were thus good candidates for NSS.

Somatic mutations of the RET proto-oncogene are not required for tumor development in multiple endocrine neoplasia type 2 (MEN 2) gene carriers.

Germ line mutations in one allele of the RET proto-oncogene predispose to the multiple endocrine neoplasia type 2 (MEN 2) syndromes. To investigate whether these inherited mutations alone can cause the development of tumors in vivo (oncogene model) or whether somatic mutations in the homologous RET allele are required for tumorigenesis (tumor suppressor gene model), we analyzed the entire coding region of both alleles of the RET gene in two MEN 2A and two MEN 2B tumors by reverse transcription-PCR and direct sequencing. No tumor-specific mutations could be detected in either allele of the RET gene in these tumors. Unlike the molecular mechanism in other hereditary tumor syndromes, somatic mutations in the homologous allele are apparently not required in MEN 2 tumorigenesis. Thus, RET genes with MEN 2-specific germ line mutations act as dominantly transforming oncogenes in vivo.

Clinical screening as compared with DNA analysis in families with multiple endocrine neoplasia type 2A.

BACKGROUND: Multiple endocrine neoplasia type 2A (MEN-2A) is characterized by medullary thyroid carcinoma in combination with pheochromocytoma and sometimes parathyroid adenoma. Missense mutations in the RET proto-oncogene are associated with MEN-2A. Their detection by DNA analysis allows the identification of carriers of the gene, in whom the risk of medullary thyroid carcinoma is 100 percent. We compared the reliability of biochemical tests with that of DNA analysis in identifying carriers of the MEN2A gene. METHODS: Starting in 1975, we screened 300 subjects in four large families with MEN-2A for expression of the disease, using measurements of plasma calcitonin after stimulation with pentagastrin or calcium and urinary excretion of catecholamines and catecholamine metabolites. We tested for carrier status by DNA analysis, including linkage analysis, and more recently by analysis of mutations in the RET gene. RESULTS: Of 80 MEN2A gene carriers (in 61 of whom carrier status was proved by DNA analysis), 66 had abnormal plasma calcitonin values and medullary thyroid carcinoma. Fourteen young carriers had normal results of plasma calcitonin tests. In 8 of these 14, thyroidectomy revealed small foci of medullary thyroid carcinoma; the remaining 6 have not yet been operated on. Of the other 220 family members, 68 were found by DNA analysis not to carry the MEN2A gene. None of these 68 subjects had medullary thyroid carcinoma or pheochromocytoma; 6 had elevated plasma calcitonin concentrations and underwent thyroidectomy but had only C-cell hyperplasia. CONCLUSIONS: Unlike biochemical tests, DNA analysis permits the unambiguous identification of MEN2A gene carriers.