Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.      

BRCA2与LIM结构域蛋白FHL2的相互作用

目的：应用酵母双杂交方法筛选BRCA2相互作用蛋白编码基因，验证其相互作用并研究其功能联系。方法：以BRCA2基因3′端片段构建酵母双杂交质粒，筛选正常人乳腺上皮细胞cDNA库，获得编码相互作用蛋白的基因，采用免疫共沉淀、哺乳细胞双杂交和荧光酶测定等方法进一步验证蛋白间相互作用和功能联系．结果：采用酵母双杂交系统筛选，获得了多个编码BRCA2相互作用蛋白的基因，其中包括已知的FHL2蛋白；免疫共沉淀和哺乳动物细胞双杂交试验显示BRCA2和FHL2在体内特异性结合，并证实FHL2在体内形成同源二聚体；转录活性分析发现BRCA2与FHL2有协同转录激活作用。结论：发现BRCA2与FHL2蛋白间相互作用和功能联系，为BRCA2功能研究提供了新的方向。     

Modalities of Empowerment and Strengthening of Ex-servicemen

Background

Older people including ex-servicemen (ESM) in India are left to fend for themselves due to nuclear family system. Most children are not staying with them due to reasons such as acquiring of education or employment at distant places. These factors, coupled with deteriorating health with advancing age, change in value system and attitude amongst children who give more importance to materialism, it was felt necessary to find modalities to empower and strengthen ESM by available methods of including progressive weight training, yoga and meditation.

Methods

An interventional “Before and After” study was planned for a period of three months for in-patient ESM of a military hospital for common lifestyle diseases namely stroke, diabetes, senile depression, acute myocardial infarction and hypertension. Data of anthropometry, laboratory investigations and Hamilton rating scale for depression (HRSD) were taken before the study. These cases were then subjected to interventions (progressive weight training and spiritual health enhancement by yoga with meditation for three months) followed by same measurements again.

Result

Statistically significant differences were observed before and after study with respect to pulse, respiratory rate, blood pressure, blood glucose, total cholesterol, low density lipoproteins (LDL), very low density lipoproteins (VLDL) and Hamilton depression score (p<0.05). The sleep pattern, sense of well being and posture while sitting to standing and walking with degree of support (stick/other person''s help) improved considerably after the interventions.

Conclusion

ESM are recommended to be trained much before retirement on progressive weight training and yoga in regimental centre/station institute. Provision to be made in existing ex-servicemen contributory health scheme (ECHS) polyclinics for employment of part time trainers for weight training and yoga. The physical and spiritual strengthening results in better capability to deal with old age and its problems.Key Words: Ex-servicemen, Progressive weight training, Yoga, Spiritual health     

Risk Factors and Hospitalization Costs of Dementia Patients: Examining Race and Gender Variations

Aims:

To examine the variation in risk factors and hospitalization costs among four elderly dementia cohorts by race and gender.

Materials and Methods:

The 2008 Tennessee Hospital Discharged database was examined. The prevalence, risk factors and cost of inpatient care of dementia were examined for individuals aged 65 years and above, across the four race gender cohorts - white males (WM), black males (BM), white females (WF), and black females (BF).

Results:

3.6% of patients hospitalized in 2008 had dementia. Dementia was higher among females than males, and higher among blacks than whites. Further, BF had higher prevalence of dementia than WF; similarly, BM had a higher prevalence of dementia than WM. Overall, six risk factors were associated with dementia for the entire sample including HTN, DM, CKD, CHF, COPD, and stroke. These risk factors varied slightly in predicting dementia by race and gender. Hospital costs were 14% higher among dementia patients compared to non-dementia patients.

Conclusions:

There exist significant race and gender disparities in prevalence of dementia. A greater degree of co-morbidity, increased duration of hospital stay, and more frequent hospitalizations, may result in a higher cost of inpatient dementia care. Aggressive management of risk factors may subsequently reduce stroke and cost of dementia care, especially in the black population. Race and gender dependent milestones for management of these risk factors should be considered.     

Respiratory distress syndrome treated with human surfactant: radiographic findings

Chest radiographs of 18 newborns treated with endotracheal instillation of human surfactant for respiratory distress syndrome (RDS) were compared with those of 18 similar but untreated infants. In the treated infants, severity of RDS significantly improved after surfactant administration. Most treated infants (16/18) exhibited a left-to-right shunt, presumably through a patent ductus arteriosus; similar findings were noted in untreated infants (17/18). Complications of respiratory assistance in the treated infants included transient pulmonary interstitial emphysema (n = 1), pneumothorax (n = 1), and mild (n = 4) to moderate (n = 2) bronchopulmonary dysplasia; the incidences of these complications did not exceed those in untreated infants. In three treated infants, a transient interstitial lung disease developed 3-4 days after surfactant administration.     

Natural Occurrence of Horizontal Transfer of Mycobacterium avium- Specific Insertion Sequence IS1245 to Mycobacterium kansasii

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. The insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.Mycobacterium avium and Mycobacterium kansasii are major human mycobacterial pathogens, and both can cause pulmonary infections in immunocompetent individuals, as well as disseminated infections in the immunocompromised (6). Coinfection with M. avium and M. kansasii has also been documented in HIV-positive patients (8, 15). Current treatment of M. avium and M. kansasii infections is based on long-term antibiotic therapy and relies on different drug combinations, justifying all efforts directed to the correct identification of clinical isolates. Insertion sequences (IS) are mobile genetic elements within mycobacteria that are often species specific, a property that can be exploited for diagnosis and epidemiological studies (4). The host range of the IS1245 element was considered to be limited to M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum (3, 9, 11-13, 17).Detection of IS1245 by PCR was used for confirmation of the presence of M. avium in a mixed culture from a bone marrow specimen from an HIV-positive patient. Fifteen slow-growing colonies were recovered from the bone marrow primary culture by plating dilutions on Middlebrook 7H10 solid medium supplemented with oleic acid, albumin, catalase, and dextrose (7H10-OADC). Four colonies (88.1 to 88.4) were nonpigmented, slow-growing mycobacteria identified as M. avium by PCR-restriction enzyme analysis (PRA) using the 16S-23S rRNA internal transcribed spacer (ITS) sequence as the target (14). Eleven colonies (88.5 to 88.15) produced yellow pigment after exposure to light and were identified as M. kansasii by PRA-ITS (Fig. ​(Fig.11 A). For further characterization of these colonies, a 427-bp fragment from IS1245 was amplified by PCR (3). Unexpectedly, IS1245 amplicons were detected not only in the nonpigmented M. avium colonies but also in 8 out of 11 M. kansasii colonies (Fig. ​(Fig.1B).1B). Amplicons generated with M. kansasii DNA were sequenced and showed 100% identity with the deposited IS1245 sequence (accession number {"type":"entrez-nucleotide","attrs":{"text":"L33879","term_id":"600521","term_text":"L33879"}}L33879) (data not shown). Final identification of the 15 colonies to the species level was obtained by sequencing a 440-bp fragment of the 5′ 16S rRNA gene (Escherichia coli positions 54 to 510) (16). Colonies 88.1 through 88.4 showed 100% sequence similarity to the corresponding sequence of M. avium type strain ATCC 25291 (accession number {"type":"entrez-nucleotide","attrs":{"text":"EF521895","term_id":"157004948","term_text":"EF521895"}}EF521895). Colonies 88.5 through 88.15 showed 100% similarity to the corresponding sequence of M. kansasii type strain CIP 104589 (accession number {"type":"entrez-nucleotide","attrs":{"text":"AF547940","term_id":"27733764","term_text":"AF547940"}}AF547940). The two sequences differed at 12 positions (97.3% similarity).Open in a separate windowFIG. 1.Molecular characterization of 15 single colonies isolated from the original M. avium-M. kansasii mixed culture by PRA-ITS (A), PCR-IS1245 (B), RFLP-IS1245 (C), and pulsed-field gel electrophoresis (D). Lanes: colonies 88.1 to 88.4, M. avium; colonies 88.5 to 88.15, M. kansasii; −, PCR negative control (water); +, PCR positive control (DNA from M. avium ATCC 25291^T). Asterisks indicate RFLP-IS1245 band polymorphisms in M. avium colonies. On the right are molecular size markers.To confirm the presence of IS1245 copies in the eight colonies of M. kansasii, restriction fragment length polymorphism (RFLP) experiments were performed by using IS1245 as a probe (17). While the M. avium colonies (88.1 to 88.4) produced multiband RFLP patterns characteristic of M. avium subsp. hominissuis, the eight M. kansasii colonies that had produced amplicons by PCR-IS1425 (88.8 to 88.15) showed a single IS1245-hybridizing band of approximately 4,750 bp. Moreover, a second IS1245-hybridizing band was detected in two of these colonies (88.11 and 88.14). Furthermore, the three PCR-IS1245-negative colonies of M. kansasii (88.5 to 88.7) lacked IS1245 hybridization bands (Fig. ​(Fig.1C).1C). Except for the presence of IS1245, pulsed-field gel electrophoresis patterns indicated that all of the 11 M. kansasii colonies were, in fact, the same strain (Fig. ​(Fig.1D1D).The results obtained strongly suggest that M. kansasii acquired the IS1245 element from the M. avium strain in a horizontal DNA transfer event which occurred either within the patient or during specimen or culture storage. This hypothesis was reinforced by the fact that both species were isolated from a unique specimen collected from one patient and by the finding of several M. kansasii colonies (88.5 to 88.7) of the same strain devoid of this insertion sequence element in that specimen (Fig. ​(Fig.1B,1B, C, and D). The presence of the IS1245 element in M. kansasii was not detected by the analysis of five additional M. avium-M. kansasii mixed cultures from different patients (data not shown), which agrees with the hypothesis that the event described here was the result of a particular horizontal DNA transfer episode.Antimicrobial susceptibility testing was performed with three isolates using a microdilution method previously described (10). The drugs tested included streptomycin, isoniazid, ethambutol, rifampin, rifabutin, ciprofloxacin, amikacin, azithromycin, and clarithromycin. Interpretative criteria followed NCCLS recommendations (10). M. avium colony 88.3 was resistant to isoniazid (MIC, 1 μg/ml), ethambutol (MIC, 8 μg/ml), azithromycin (MIC, >8 μg/ml), and clarithromycin (MIC, >32 μg/ml). M. kansasii colonies 88.5 (IS1245 negative) and 88.8 (IS1245 positive) were susceptible to all of the drugs tested, showing that the acquisition of IS1245 did not change the pattern of susceptibility of this M. kansasii strain to the drugs tested.In order to examine the stability of IS1245 in M. kansasii, two colonies (88.11 and 88.12) were grown in liquid Middlebrook 7H9-OADC on a shaker at 37°C until the optical density at 600 nm reached 0.6 to 0.8. Ten serial passages were then performed by diluting the cultures 1:10 in the same medium and incubating them again under the same conditions. After 10 passages, the cultures were added to solid 7H10-OADC by the spread plate method and 19 isolated colonies were analyzed by RFLP-IS1245. The original IS1245 hybridization bands were maintained in all of the colonies isolated after 10 passages, demonstrating the stability of this insertion sequence in M. kansasii, at least during short-term (4 months) in vitro processing. However, one to three new hybridization bands were visualized in 11 out of 19 M. kansasii colonies analyzed, pointing to the occurrence of insertion sequence transposition by a replicative mechanism (Fig. ​(Fig.22).Open in a separate windowFIG. 2.RFLP-IS1245 of colonies 88.11 and 88.12 and individual colonies isolated after 10 serial passages in 7H9-OADC liquid medium. Lanes: 1, original colony 88.11; 2 to 10, isolated colonies after subculture of colony 88.11; 11, original colony 88.12; 12 to 21, isolated colonies after subculture of colony 88.12. Black arrows indicate the hybridization bands present in the original colonies. On the right are molecular size markers.Colonies of both species showed variations in RFLP-IS1245 patterns during this study. Besides the IS1245 transposition detected in M. kansasii, two or three RFLP-IS1245 band polymorphisms were also detected in the M. avium colonies (Fig. ​(Fig.1C).1C). Recent IS1245 transposition events in M. avium have been observed in other studies with single colonies of the same isolate or isolates collected from individual patients over time (1, 3, 11, 12).The IS1245 insertion sequence was initially considered to be species specific for M. avium, but additional studies have shown that this element is sporadically present in M. intracellulare, M. malmoense, M. scrofulaceum, and M. nonchromogenicum (2, 7, 9). To our knowledge, the results obtained in this study confirm, for the first time, the presence of one or more copies of IS1245 in a strain of M. kansasii by PCR, RFLP, and DNA sequencing.As a consequence, the utilization of IS1245 as a genetic marker for the identification of M. avium and its distinction from M. kansasii should be done with caution and combined with other genetic markers. The absence of IS1245 copies in some M. avium strains must also be taken into account (5, 11, 13).IS1245 transposition events such as those described here can produce genome plasticity by the interruption or deletion of genes, affecting biological properties, which deserves further investigation.