Identification of Endoglin as an epigenetically regulated tumour-suppressor gene in lung cancer.

BACKGROUND: The transforming growth factor-beta (TGF- ß) pathway has been implicated in proliferation, migration and invasion of various cancers. Endoglin is a TGF-ß accessory receptor that modulates signalling. We identified Endoglin as an epigenetically silenced tumour-suppressor gene in lung cancer by means of a genome-wide screening approach, then sought to characterise its effect on lung cancer progression. METHODS: Methylation microarray and RNA sequencing were carried out on lung cancer cell lines. Epigenetic silencing of Endoglin was confirmed by methylation and expression analyses. An expression vector and a 20-gene expression panel were used to evaluate Endoglin function. Pyrosequencing was carried out on two independent cohorts comprising 112 and 202 NSCLC cases, respectively, and the impact of Endoglin methylation on overall survival (OS) was evaluated. RESULTS: Methylation in the promoter region resulted in silencing of Endoglin, which could be reactivated by demethylation. Increased invasion coupled with altered EMT marker expression was observed in cell lines with an epithelial-like, but not those with a mesenchymal-like, profile when Endoglin was absent. Methylation was associated with decreased OS in stage I but not in stages II-III disease. CONCLUSIONS: We show that Endoglin is a common target of epigenetic silencing in lung cancer. We reveal a link between Endoglin silencing and EMT progression that might be associated with decreased survival in stage I disease.

Mutations in GDI1 are responsible for X-linked non-specific mental retardation.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes uGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

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.

Methylation of the calcium channel regulatory subunit α2δ-3 (CACNA2D3) predicts site-specific relapse in oestrogen receptor-positive primary breast carcinomas.

BACKGROUND: Calcium is an important intracellular messenger that mediates many biological processes that are relevant to the malignant process. Calcium ion channels are key in controlling the intracellular calcium, and little is known about their role in human cancer. METHODS: We used qPCR and pyrosequencing to investigate expression and epigenetic regulation of the calcium channel regulatory subunit α(2)δ-3 (CACNA2D3) in breast cancer cell lines, primary cancers and metastatic lesions. RESULTS: Expression of CACNA2D3 mRNA is regulated in breast cancer cell lines by methylation in the CpG island located in the 5' regulatory region of the gene. Expression is upregulated by azacytidine (AZA) in cells with CpG island methylation but unaffected in cells lacking methylation. In primary breast carcinomas, methylation is more common in cancers, which subsequently relapse with loco-regional and, particularly, visceral metastatic disease in both oestrogen receptor-α (ER)-positive and -negative cases. Furthermore, CACNA2D3 CpG island is frequently methylated in breast cancer that has metastasised to the central nervous system. CONCLUSION: Methylation-dependent transcriptional silencing of CACNA2D3 may contribute to the metastatic phenotype of breast cancer. Analysis of methylation in the CACNA2D3 CpG island may have potential as a biomarker for risk of development of metastatic disease.

High frequency of complex TP53 mutations in CNS metastases from breast cancer.

BACKGROUND: Brain metastasis from breast cancer is usually associated with a poor prognosis and early death. Alteration of p53 may contribute to malignant progression by abrogation of apoptosis induced by oncogene activation and by acquisition of gain-of-function properties, which promote tumour aggression. Mutation in TP53 occurs at high frequency in carcinomas of the lung and gastro-intestinal tract, but is much less frequent, at 25%, in primary breast cancer. The frequency of TP53 alteration in the central nervous system (CNS) metastatic breast cancer is not known. METHODS: In all, 23 cases of histologically confirmed CNS metastatic breast cancer were identified and the coding sequence of TP53 determined. TP53 was also sequenced in two control series of primary breast carcinomas from independent clinical centres. RESULTS: We demonstrate a strikingly high frequency of TP53 mutation in the CNS metastatic lesions with an over-representation of complex mutations (non-sense/deletions/insertions). Complex mutations occur in metastatic lesions in both triple-negative breast cancer and hormone receptor/HER2-positive cases. Analysis of paired primary carcinomas and brain metastatic lesions revealed evidence for both clonal selection and generation of new mutations (missense and complex) in progression from a primary breast carcinoma to brain metastasis. CONCLUSION: Mutation in TP53 is the most common genetic alteration reported during metastasis to the brain in breast cancer.

Site-specific CpG methylation in the CCAAT/enhancer binding protein delta (CEBPδ) CpG island in breast cancer is associated with metastatic relapse.

BACKGROUND: The CCAAT/enhancer binding protein delta (CEBPδ) is a member of a highly conserved family of basic region leucine zipper transcription factors. It has properties consistent with a tumour suppressor; however, other data suggest that CEBPδ may be involved in the metastatic process. METHODS: We analysed the expression of CEBPδ and the methylation status of the CpG island in human breast cancer cell lines, in 107 archival cases of primary breast cancer and in two series of metastatic breast cancers using qPCR and pyrosequencing. RESULTS: Expression of CEBPδ is downregulated in primary breast cancer by site-specific methylation in the CEBPδ CpG island. Expression is also downregulated in 50% of cases during progression from primary carcinoma to metastatic lesions. The CEBPδ CpG island is methylated in 81% metastatic breast cancer lesions, while methylation in the CEBPδ CpG island in primary cancers is associated with increased risk of relapse and metastasis. CONCLUSION: CCAAT/enhancer binding protein delta CpG island methylation is associated with metastasis in breast cancer. Detection of methylated CEBPδ genomic DNA may have utility as an epigenetic biomarker of primary breast carcinomas at increased risk of relapse and metastasis.

NT5E CpG island methylation is a favourable breast cancer biomarker.

BACKGROUND: Relapse risk assessment and individual treatment recommendations remain suboptimal for breast cancer patients. In the light of existing preclinical and clinical data, we studied NT5E (5'-nucleotidase, ecto) expression and NT5E CpG island methylation in breast cancer. METHODS: We used RT-PCR, qPCR, methylation-specific PCR and pyrosequencing to analyse NT5E in breast carcinoma cell lines and primary and breast carcinomas. RESULTS: NT5E CpG island methylation was inversely associated with NT5E expression in breast carcinoma cell lines. In clinical series, patients whose primary tumours had NT5E CpG island methylation were less likely to develop metastasis (P=0.003, OR=0.34, 95% CI: 0.17-0.69). In 3/4 paired samples, NT5E was methylated in primary tumours and demethylated in CNS metastases. Patients progressing to non-visceral as compared with visceral metastases were more likely to have NT5E CpG island methylation in primary tumours (P=0.01, OR=11.8). Patients with tumours lacking detectable methylation had shorter disease-free survival (DFS) (P=0.001, HR=2.7) and overall survival (OS) (P=0.001, HR=3). The favourable prognostic value of NT5E methylation was confirmed in oestrogen receptor negative (P=0.011, HR=3.27, 95% CI: 1.31-8.12) and in triple negative cases (P=0.004; HR=6.2, 95% CI: 1.9-20). Moreover, we observed a more favourable outcome to adjuvant chemotherapy in patients whose tumours were positive for NT5E CpG island methylation: DFS (P=0.0016, HR=5.1, 95% CI: 1.8-14.37) and OS (P=0.0005, HR=7.4, 95% CI: 2.416-23.08). CONCLUSION: NT5E CpG island methylation is a promising breast cancer biomarker.

The collagen prolyl hydroxylases are novel transcriptionally silenced genes in lymphoma.

BACKGROUND: Prolyl hydroxylation is a post-translational modification that affects the structure, stability and function of proteins including collagen by catalysing hydroxylation of proline to hydroxyproline through action of collagen prolyl hydroxylases3 (C-P3H) and 4 (C-P4H). Three C-P3Hs (nomenclature was amended according to approval by the HGNC symbols and names at http://www.genenames.org/ and Entrez database at http://www.ncbi.nlm.nih.gov/gene) leucineproline-enriched proteoglycan (leprecan) 1 (Lepre1), leprecan-like 1 (Leprel1), leprecan-like 2 (Leprel2) and two paralogs Cartilage-Related Protein (CRTAP) and leprecan-like 4 (Leprel4) are found in humans. The C-P4Hs are tetrameric proteins comprising a variable α subunit, encoded by the P4HA1, P4HA2 and P4HA3 genes and a constant ß subunit encoded by P4HB. METHODS: We used RT-PCR, qPCR, pyrosequencing, methylation-specific PCR, western blotting and immunohistochemistry to investigate expression and regulation of the C-P3H and C-P4H genes in B lymphomas and normal bone marrow. RESULTS: C-P3H and C-P4H are downregulated in lymphoma. Down-regulation is associated with methylation in the CpG islands and is detected in almost all common types of B-cell lymphoma, but the CpG islands are unmethylated or methylated at lower levels in DNA isolated from normal bone marrow and lymphoblastoid cell lines. Methylation of multiple C-P3H and C-P4H genes is present in some lymphomas, particularly Burkitt's lymphoma. CONCLUSIONS: Methylation of C-P3H and C-P4H is common in B lymphomas and may have utility in differentiating disease subtypes.

Surgical treatment of thymoma: personal experience.

INTRODUCTION: Thymomas (THs) are rare epithelial tumors of the thymus gland. In this study we report our personal experience in the management and surgical treatment of THs. CASE REPORTS: We report two clinical cases treated with combined therapy (surgery followed by adjuvant therapy). RESULTS: Total transternal thymectomy was performed in both patients. The post-operative course was uneventful. The patients received adjuvant radiotherapy and chemotherapy. No relapse has been observed during follow-up. DISCUSSION: THs are usually slowly growing tumors with similar incidence in both sexes. They occur through a wide age range, with a peak in the fifth and sixth decades. Distinctive features reminiscent of the normal thymus make the pathologic diagnosis of THs easy in most cases. Malignant behaviour is indicated by microscopic or macroscopic invasion of the tumor capsule or surrounding organs or by the presence of metastases. Although there is no standardized staging system for thymoma, the one proposed by Masaoka is commonly employed. Total thymectomy is the procedure of choice, even for encapsulated tumors, with carefully exploration of the mediastinum for evidence of ectopic thymic tissue or local invasion. CONCLUSIONS: Despite an indolent course and a cytologically bland appearance, all thymic tumors can manifest a malignant behavior. Surgery continues to be the mainstay of treatment, and the ability to achieve complete resection seems to be the most important prognostic factor. Multimodality treatment involving postoperative chemotherapy and radiotherapy appears to increase the rate of complete resection and improves survival in advanced THs.

Bilateral lung and liver hydatid cysts. Case report.

Introduction. Synchronous occurrence of pulmonary and hepatic hydatid cysts is an uncommon manifestation of hydatid disease that is observed in less than 10% of cases. We report a rare case of bilateral lung (with bronchial fistula) and liver cyst, surgically treated after medical therapy. Case report. A 44-year-old housewife reporting fever, anorexia and fatigue that had been present for the previous 20 days received diagnosis of bilateral lung and liver hydatid cyst. Because of the dimensions of right lung cyst and the successive bronchial fistolization, we proceeded to three-stage operation of two thoracotomies and a laparotomy to control the risk of further rupture. After surgery, all post-operatives were uneventful. Complete resolution of the therapy with no evidence of recurrence at 2 years follow-up. Conclusion. We emphasize the need to search for additional hydatids in patients who present with either pulmonary or liver hydatids. The simultaneous treatment of liver and lung should be reserved to patients in good conditions; in all other cases, especially when one cyst is more symptomatic than the others or has more risk of rupture, we prefer to treat single cyst.

Cisplatin-based chemoradiation plus cetuximab in locally advanced head and neck cancer: a phase II clinical study.

BACKGROUND: Intensification of chemoradiation for advanced head and neck squamous cell carcinoma (HNSCC) is unlikely due to toxicity. Cetuximab combined either with radiotherapy or with chemotherapy showed favourable toxic profile with positive results in both combinations. Therefore, cetuximab could intensify chemoradiation without worsening toxicity. We conducted a phase II study of chemoradiation and cetuximab. PATIENTS AND METHODS: Eligible patients had stage III-IV M0 HNSCC. Treatment consisted of three cycles of cisplatin (20 mg/m(2)/day × 5 days) and fluorouracil (200 mg/m(2)/day × 5 days) rapidly alternated to three split courses of radiotherapy up to 70 Gy and concurrent weekly cetuximab. The primary end point of the study was complete response (CR) rate. Secondary end points were toxicity, progression-free survival (PFS) and overall survival (OS). RESULTS: Fourty-five patients were enrolled: median age was 56 years, 38 had stage IV disease and 40 nodal involvement. CR occurred in 32 patients (71%). PFS and OS was 21+ months and 32.6+, respectively. Acute grade 3-4 toxic effects were in the expected range, but grade 3 radiodermatitis occurred in 33 patients. CONCLUSIONS: The combination of cetuximab, cisplatin, fluorouracil and radiotherapy leads to a very high proportion of CR and it is feasible with toxic effects similar to those expected by radiochemotherapy. The only unexpected toxicity was skin toxicity: grade 3 radiodermatitis occurred in 73% of the patients.

Non-recurrent laryngeal nerve coexisting with ipsilateral recurrent nerve: personal experience and literature review.

INTRODUCTION: Non-recurrence and variations in ascending course of the recurrent laryngeal nerve (RLN) represent a risk factor for nerve injuries during thyroid surgery. Non-recurrent laryngeal nerve (NRLN) coexisting to recurrent nerve branch is a rare anatomic anomaly. It could be a cause of nerve injuries during thyroidectomy. A systematic intraoperative nerve identification may allow an effectiveness prevention of iatrogenic injuries. CASE REPORT: We report one case of a young woman underwent to total thyroidectomy (TT) for papillary thyroid carcinoma (PTC) where we found a rare variation of the right inferior laryngeal nerve anatomy. We identified both right laryngeal nerve structures before completing thyroidectomy avoiding possible nerve damage. The postoperative course was without complications. DISCUSSION: Iatrogenic injury of RLN is one of the most serious complication in thyroid surgery. Several risk factors favouring this complication were found as the presence of anatomic variations of the inferior laryngeal nerve. Identification of a normal caliber recurrent nerve can allow the surgeon to complete the thyroid excision; diversely, in case of a smaller caliber nerve in the usual recurrent course, a careful dissection should be continued to demonstrate a possible merger with ipsilateral non-recurrent nerve. CONCLUSIONS: The aim of this paper is to report a rare case of NRLN associated to a smaller caliber branch of RNL. We emphasize that careful dissection and intimate knowledge of normal and anomaly anatomy allow for avoidance of nerve injury during surgery in the neck.

[Endoscopic rubber band ligation in treatment of esophageal varices bleeding. Personal experience]. / La legatura elastica nel trattamento endoscopico della emorragia da rottura di varici esofagee. Esperienza di un singolo centro.

INTRODUCTION: Bleeding esophageal varices is the most serious complication of the portal hypertension, and the greater cause of dead (25% of the patients). The survival after esophageal varices bleeding depends in wide part from the swiftness and effectiveness of hemostasis and from the degree of functional liver reserve. Aim of our manuscript is to report our experience about hemostasis bleeding esophageal varices with endoscopic rubber band ligation. PATIENTS AND METHODS: From January 1999 to January 2008 we performed 302 esofagogastroduodenoscopy (EGDS) for esophageal varices bleeding (M: F ratio = 1.4:1, mean age 56.4 years, 62% of cases with HCV-related cirrhosis, 29% alcoholic cirrhosis and 9% cryptogenic cirrhosis; 20% suffered from chronic renal failure, 15% diabetes mellitus, 10% hepatocellular carcinoma on cirrhosis, 5% systemic encephalopathy and 1% AIDS). RESULTS; All patients were treated within 6 hours after the first reported episode of haematemesis and all received beta-blocker therapy after the episode. In the first phase of our experience were used rechargeable elastic ligator and then multibyte, even in combination with polidocanol sclerotherapy (8%) or injection of cyanoacrylate (5%). The best results were achieved with band ligation, in terms of primitive haemostasis, rebleeding, (3%), intraoperative mortality (1%) and 6 weeks mortality (1%). CONCLUSION: To date, no single method applicable to all patients with bleeding esophageal varices, but endoscopic rubber band ligation is currently considered the first-line treatment of proper multidisciplinary approach to the patient, both during the acute event than prevention of rebleeding, because it is an effective, safe and repeatable, in experienced hands.

Neck node dissection in thyroid cancer. A review.

INTRODUCTION: thyroid cancer recurs most commonly in one or more cervical lymph nodes. Surgical treatment for differentiated or medullary thyroid carcinoma consists of total thyroidectomy (TT). The aim is to elucidate the potential benefits and drawbacks of neck dissection TT related. MATERIALS AND METHODS: differences between therapeutic and prophylactic neck dissection were analysed to prevent post-operative morbidity, neck recurrences and improve survival. DISCUSSION: there is considerable controversy regarding the treatment of patients with cervical node metastases specially in differentiated thyroid cancer. Considering that a neck dissection might help to reduce local recurrence, especially in medullary carcinoma, controversial remains regarding the modality and extension of cervical dissection. There are several surgical strategies to cervical lymphadenectomy as a prophylactic node dissection or a dissection only in symptomatic patients or the "node-picking" technique for selective lymphadenectomy or sentinel node biopsy. So it is possible to employ several kind of neck-node compartment related dissection. The risk of iatrogenic lesion during neck dissection is relatively high specially for nerve structures (i.e.: recurrent laryngeal nerve or spinal accessory nerve), so an experienced surgeon must mind the risk is higher during a re-operation in an anatomical subverted region. CONCLUSIONS: the extent of dissection and the experience of the surgeon both play important roles in determining the risk of surgical complications and recurrence. The decision to add neck dissection to total thyroidectomy weighed against documented benefits and risks. Injuries may also occur as a result of inadequate technique or as a result of poor expertise of the surgeon. We believe that deep knowledge of the thyroid region's surgical anatomy is necessary to realize a skilled and careful surgery of the neck.

Refinement of the SPG9 locus on chromosome 10q23.3-24.2 and exclusion of candidate genes.

BACKGROUND AND PURPOSE: The hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders, characterized by a progressive spasticity of the lower limbs. So far, 33 different loci (SPGs) have been mapped and the 15 genes responsible have been identified. We mapped a locus responsible for a form of spastic paraplegia, complicated by bilateral cataracts, gastroesophageal reflux with persisting vomiting and amyotrophy to chromosome 10q23.3-q24.2, in an Italian family. The critical region was in a 12 cm chromosomal interval between markers D10S564 and D10S603 (SPG9, MIM601162). In the same region, two other forms of HSP have been recently mapped: SPG27 and SPG33. In the latter case, the gene responsible has been identified. MATERIALS AND METHODS: To better characterize this region, we genotyped individuals from SPG9-linked families using additional markers and reduced the candidate region to a 4.8 Mb, excluding several genes by positional cloning. RESULTS: The refined SPG9 locus is positioned completely within SPG27 and does not include the SPG33 gene. DISCUSSION: Fifty-two transcripts are present in the refined critical region and 25 strong candidates have been excluded as disease causing genes by direct sequencing. Six of them were also excluded as responsible for SPG27.

FRAXE mutation in a mentally retarded subject and in his phenotypically normal twin brother.

The FRAXE fragile site, 600 kb distal to the more common FRAXA, has been reported to be expressed in subjects with mild non-syndromal mental retardation (MR). Amplification of more than 200 GCC repeats, associated with methylation of the adjacent CpG island at Xq28, leads to the expression of the fragile site. In 1996 a large gene, FMR2, transcribed distally from the CpG island and downregulated by repeat expansion and methylation, was identified. Among 232 mentally retarded patients, tested FRAXA negative, we identified an Italian family segregating a hypermethylated expansion at the FRAXE locus in two dizygotic twin brothers, their sister and their mother. The index case was referred at 23 years of age with severe MR, epilepsy, a dysmorphic face with a high arched palate, marfanoid habitus and hyperreflexia of the lower limbs. His brother was referred to as normal and psychometric tests confirmed he is not mentally retarded. All members of the family underwent FRAXE molecular analysis, after cytogenetic expression of the fraX site and negative FRAXA test. Interestingly, an expansion and a hypermethylation at the FRAXE locus were found in all of them. Fibroblasts from the clinically normal brother were assayed for FMR2 expression and the transcription of the gene was found to be silenced. The presence of a phenotypically normal male with absent FMR2 expression in fibroblasts suggests that the relationship between the FRAXE mutation, FMR2 expression and MR needs to be further investigated.

A refined physical and transcriptional map of the SPG9 locus on 10q23.3-q24.2.

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous disorder characterised by progressive spasticity of the lower limbs. Beside 'pure' forms of HSP, 'complicated' forms are reported, where spasticity occurs associated with additional symptoms. We recently described an Italian family with a complicated dominant form of HSP (SPG9) and we mapped the gene responsible to 10q23.3-q24.2, in a 12cM interval between markers D10S564 and D10S603. The phenotypic manifestations in our family are reminiscent of those already described in a smaller British pedigree. We typed individuals from this British family using markers located in the SPG9 critical interval and haplotype reconstruction showed the disorder co-segregating with SPG9. To characterise the SPG9 region better, we constructed a contig of 22 YACs, assigned it to 18 polymorphic markers and positioned 54 ESTs. Furthermore, we searched for ESTs containing a trinucleotide repeat sequence, since anticipation of symptoms was reported in both families. Finally, analysis of a muscle biopsy specimen from one patient was normal, suggesting that, contrary to SPG7, mitochondrial disturbance could not be a primary feature of SPG9.

Identification and characterization of a novel human brain-specific gene, homologous to S. scrofa tmp83.5, in the chromosome 10q24 critical region for temporal lobe epilepsy and spastic paraplegia.

We describe the structure, genomic organization, and some transcription features of a human brain-specific gene previously localized to the genomic region involved in temporal lobe epilepsy and spastic paraplegia on chromosome 10q24. The gene, which consists of six exons disseminated over 16 kb of genomic DNA, is highly homologous to the porcine tmp83.5 gene and encodes a putative transmembrane protein of 141 amino acids. Unlike its porcine homolog, from which two mRNAs with different 5'-sequences are transcribed, the human gene apparently encodes three mRNA species with 3'-untranslated regions of different sizes. Mutation analysis of its coding sequence in families affected with temporal lobe epilepsy or spastic paraplegia linked to 10q24 do not support the involvement of this gene in either diseases.

Molecular and cytogenetic analysis of the fragile X syndrome in a series of 453 mentally retarded subjects: a study of 87 families.

We report on a series of 453 mentally retarded subjects investigated for fragile X syndrome from 1982 to July 1995. The 22% rate of efficiency of FRAX positivity indicated a significant preselection by the clinicians. However, this rate dropped to 11% in the last year. Since 1992, Southern blot analysis was extended to include family members of the 87 positive subjects, for a total of 442 individuals examined with the probe StB12.3. In addition to premutated (118), fully mutated (148), and pre/full mutation mosaic subjects (27), 14 atypical cases were found. Some of these cases are described in more detail. In particular, we report on the hybridization and polymerase chain reaction data of 2 fragile X subjects with full mutation and a 2.8-kb allele and 1 with full mutation and a 2.4-kb allele. An intellectually normal male with 18% of fraXq27.3 and an unmethylated full mutation is also described. Finally, a mentally retarded child with only a lower allele of 2.7 kb is presented.

Mosaicism for the full mutation and a microdeletion involving the CGG repeat and flanking sequences in the FMR1 gene in eight fragile X patients.

The molecular mechanism of the fragile X syndrome is based on the expansion of an unstable CGG repeat in the 5' untranslated region of the FMR1 gene in most patients. This expansion is associated with an abnormal DNA methylation leading to the absence of production of FMR1 protein (FMRP). Such expansion apparently predisposes the repeat and flanking regions to further instability that may lead to mosaic conditions with a full mutation and a premutation or, rarely, with normal or reduced alleles that can sometimes be transcriptionally active. In this study we describe eight unrelated fragile X patients who are mosaic for both a full mutation and an allele of normal (four cases) or reduced size (four cases). Sequencing analysis of the deletion breakpoints in 6 patients demonstrated an internal deletion confined to the CGG repeat in four of them, which represents the most likely explanation for the regression of the full mutation to a normal sized allele. In two patients with a reduced allele, the deletion encompassed the entire CGG repeat and part of the flanking regions. Analysis of FMRP by Western blot was performed in one of the mosaics with a normal sized allele and in three of those with a reduced allele. In the first patient's lymphocytes FMRP was detected, whereas in the three other patients the deletion is likely to impair transcription as no FMRP was present in their lymphocytes.