An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis.

(CGG)(n) repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel, single-tube CGG repeat primed FMR1 PCR technology was designed with two gene-specific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG)(n) repeat. This PCR was evaluated with 171 unique DNA samples, including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis, including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore, a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles, was resolved with 100% accuracy. Last, AGG interrupter sequences, which may influence the risk of (CGG)(n) expansion in the children of some carriers, were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result, this PCR provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods.

High-risk fragile x screening in Guatemala: use of a new blood spot polymerase chain reaction technique.

BACKGROUND: Because fragile X syndrome (FXS) is prevalent, it has become the subject of newborn and high-risk screening efforts. International screening, however, can be financially and logistically prohibitive, particularly in countries where resources may be scarce. Recently, we have developed a screening test on blood spot that can detect expanded alleles from the normal through the full mutation range in both males and females. It is accurate, rapid, inexpensive, and applicable on blood spots and therefore ideal for international screening. The use of this blood spot screening technique was piloted in "a high-risk screening" study of individuals in Guatemala. METHODS: One hundred and five blood spots from subjects from Guatemala were screened for the Fragile X Mental Retardation 1 mutation. They were classified as "high-risk" through placement into one of the following five categories: (a) relatives of someone with a previous FXS diagnosis, (b) individuals with confirmed autism, (c) individuals with confirmed intellectual disability, (d) individuals with Parkinson's-like presentation, and (e) individuals with a family history of intellectual disability but no confirmed cases of FXS. RESULTS: Fifteen of the individuals tested yielded an expanded allele, 10 premutations and 5 full mutations. All 15 expansions were found in individuals with a relative with a confirmed FXS diagnosis. No expansions were found in the other clinical groups. CONCLUSIONS: Blood spot polymerase chain reaction screening is an effective, cost-efficient method to conduct cascade testing in families with a known history of FXS, even in small screening cohorts.

Expansion of an FMR1 grey-zone allele to a full mutation in two generations.

Fragile X Syndrome is caused by the expansion of an unstable CGG-repeat tract in the 5'-UTR of the FMR1 gene, which generally results in transcriptional silencing and consequent absence of the FMR1 protein. To date, the smallest premutation allele reported to expand to a full mutation allele in a single generation is 59 CGG repeats. Here, we report a single-generation expansion to a full mutation allele (male with approximately 538 CCG repeats) from a mother who is a carrier of a premutation allele of 56 CGG repeats. Furthermore, the maternal grandfather was a carrier of a gray (or intermediate)-zone allele (45 to 54 repeats) of 52 CGG repeats. Thus, in this family, a gray-zone allele expanded to the full mutation range in two generations. Interestingly, the two AGG interruptions present in the grandfather's allele were absent in the mother's premutation allele. These observations underscore the need to consider carriers of alleles of greater than 55 CGG repeats as being at risk for transmission of a full mutation allele in a single generation, and those with even smaller alleles in the gray zone as being at risk of having grandchildren with full mutation alleles.

Screening for expanded alleles of the FMR1 gene in blood spots from newborn males in a Spanish population.

Fragile X syndrome, which is caused by expanded CGG repeats of the FMR1 gene, is associated with a broad spectrum of clinical involvement and is the most common inherited form of intellectual disability. Early diagnosis and intervention are likely to lead to improved outcome for children with fragile X syndrome, but such strategies require better estimates of the frequencies of expanded alleles of the FMR1 gene. In this study, we report the results of a newborn screening study of 5267 male blood spots collected from the Northwest region of Spain as part of the national newborn screening program. The blood spots were screened using a rapid polymerase chain reaction-based method that is capable of identifying the presence of all expanded alleles for both males and females. The screened samples included 199 gray zone alleles, 21 premutation alleles, and two full mutation alleles (1 in 2633). The frequency of premutation alleles was three times higher (1 in 251) than the quoted value of 1 in 813 from a Canadian population and is fully consistent with the results of large-scale Israeli screening studies. Our results demonstrate that newborn screening for the presence of expanded FMR1 alleles is an effective means for defining the distribution of expanded FMR1 alleles in newborn populations; as such, this method is suitable for large-scale newborn screening.

Screening for the presence of FMR1 premutation alleles in women with parkinsonism.

BACKGROUND: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive, late-onset neurodegenerative disease that affects older carriers of premutation (CGG) repeat expansions of the fragile X mental retardation 1 (FMR1) gene. Clinical features include intention tremor, gait ataxia, memory loss, peripheral neuropathy, autonomic dysfunction, and parkinsonism. The presence of parkinsonism in FXTAS raises the possibility that some individuals who have Parkinson disease are actually carriers of a premutation FMR1 allele. OBJECTIVE: To screen DNA samples from a large cohort of females with Parkinson disease for an excess of expanded alleles of the FMR1 gene. DESIGN AND PATIENTS: We screened a cohort of 595 women with parkinsonism, the largest screening of a parkinsonism-associated group to date, for the presence of an FMR1 premutation allele (55-200 CGG repeats). The screening protocol uses an enhanced polymerase chain reaction method capable of flagging any FMR1 expanded CGG repeat in women as well as in men. SETTING: Diagnostic assessments were performed at an outpatient tertiary clinic (Parkinson Institute, Milan). Genotyping was conducted at the University of California, Davis. MAIN OUTCOME MEASURES: CGG repeat number and clinical/neuroimaging assessments of patients with Parkinson disease were conducted. Two premutation carriers were identified. RESULTS: Two individuals possessed an FMR1 allele in the premutation range (CGG repeats: 30 and 75; 30 and 115). This carrier frequency (2 of 595 [0.34%]) is not significantly different from estimates of the allele frequency among women in the general population (0.4%-0.8%). Clinical and radiologic features of these 2 patients were similar to those of the general Parkinson disease population; however, 1 patient (115 CGG repeats) had a family history of 2 sons with the fragile X syndrome. CONCLUSION: Screening of women within the parkinsonism clinical spectrum is unlikely to be productive in the absence of additional medical or family history suggestive of involvement of the FMR1 gene.

Expanded clinical phenotype of women with the FMR1 premutation.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is generally considered to be uncommon in older female carriers of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene; however, neither prevalence, nor the nature of the clinical phenotype, has been well characterized in female carriers. In this study, we evaluated 146 female carriers (mean, 42.3 years; range, 20-75 years) with and without core features of FXTAS (tremor; gait ataxia), and 69 age-matched controls (mean, 45.8 years; range, 21-78 years). Compared with controls, carriers with definite or probable FXTAS had greater medical co-morbidity, with increased prevalence of thyroid disease (P = 0.0096), hypertension (P = 0.0020), seizures (P = 0.0077), peripheral neuropathy (P = 0.0040), and fibromyalgia (P = 0.0097), in addition to the typical symptoms of FXTAS-tremor (P < 0.0001) and ataxia (P < 0.0001). The non-FXTAS premutation group had more complaints of chronic muscle pain (P = 0.0097), persistent paraesthesias in extremities (P < 0.0001), and history of tremor (P < 0.0123) than controls. The spectrum of clinical involvement in female carriers with FXTAS is quite broad, encompassing a number of medical co-morbidities as well as the core movement disorder. The remarkable degree of thyroid dysfunction (17% in the non-FXTAS group and 50% in the FXTAS group) warrants consideration of thyroid function studies in all female premutation carriers, particularly those with core features of FXTAS.

A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.

Fragile X syndrome, the most common inherited cause of intellectual impairment and the most common single gene associated with autism, generally occurs for fragile X mental retardation 1 (FMR1) alleles that exceed 200 CGG repeats (full-mutation range). Currently, there are no unbiased estimates of the number of full-mutation FMR1 alleles in the general population; a major obstacle is the lack of an effective screening tool for expanded FMR1 alleles in large populations. We have developed a rapid polymerase chain reaction (PCR)-based screening tool for expanded FMR1 alleles. The method utilizes a chimeric PCR primer that targets randomly within the expanded CGG region, such that the presence of a broad distribution of PCR products represents a positive result for an expanded allele. The method is applicable for screening both males and females and for allele sizes throughout the premutation (55 to 200 CGG repeats) and full-mutation ranges. Furthermore, the method is capable of rapid detection of expanded alleles using as little as 1% of the DNA from a single dried blood spot. The methodology presented in this work is suitable for screening large populations of newborn or those at high risk (eg, autism, premature ovarian failure, ataxia, dementia) for expanded FMR1 alleles. The test described herein costs less than $5 per sample for materials; with suitable scale-up and automation, the cost should approach $1 per sample.

A novel 165-kDa Golgin protein induced by brain ischemia and phosphorylated by Akt protects against apoptosis.

A cDNA encoding a novel protein was cloned from ischemic rat brain and found to be homologous to testis Mea-2 Golgi-associated protein (Golga3). The sequence predicted a 165-kDa protein, and in vitro translated protein exhibited a molecular mass of 165-170 kDa. Because brain ischemia induced the mRNA, and the protein localized to the Golgi apparatus, this protein was designated Ischemia-Inducible Golgin Protein 165 (IIGP165). In HeLa cells, serum and glucose deprivation-induced caspase-dependent cleavage of the IIGP165 protein, after which the IIGP165 fragments translocated to the nucleus. The C-terminus of IIGP165, which contains a LXXLL motif, appears to function as a transcriptional co-regulator. Akt co-localizes with IIGP165 protein in the Golgi in vivo, and phosphorylates IIGP165 on serine residues 345 and 134. Though transfection of IIGP165 cDNA alone does not protect HeLa cells from serum deprivation or Brefeldin-A-triggered cell death, co-transfection of both Akt and IIGP165 cDNA or combined IIGP165-transfection with PDGF treatment significantly protects HeLa cells better than either treatment alone. These data show that Akt phosphorylation of IIGP165 protects against apoptotic cell death, and add to evidence that the Golgi apparatus also plays a role in regulating apoptosis.

[Clinicopathologic features and treatment of ductal carcinoma in situ of the breast].

OBJECTIVE: To investigate the clinicopathologic features of and appropriate treatment of ductal carcinoma in situ of the breast (DCIS). METHODS: The clinical and pathologic data of 41 cases of DCIS, aged 52.7 (30-82), 15 of which were diagnosed as with ductal carcinoma in situ with microinvasion (DCIS-MI) and of which 18 were in the Van Nuys grade I, 13 in the grade II, and 10 in the grade III, were collected and analyzed. Immunohistochemical analysis was performed to examine the expressions of estrogen receptor (ER), progesterone receptor (PR), proliferating cell nuclear antigen (PCNA), P53 and C-erbB-2. RESULTS: Microinvasion was correlated with the histologic categories (chi(2) = 4.60, P < 0.05) and tumor size (chi(2) = 9.78, P < 0.05) significantly. The expression rates of ER, PR, PCNA, P53, and C-erbB-2 were 68.3%, 65.9%, 63.4%, 26.8%, and 46.3%, respectively. There was no significant difference in the expression of the biologic markers between the patients with DCIS and those with DCIS-MI. The expression levels of ER, P53, and C-erbB-2 were correlated with the histologic categories significantly (chi(2) = 11.45, 11.97, 4.38, P < 0.05). CONCLUSION: The Van Nuys histologic classification accords with the requirement of clinical treatment and prognosis. The patients with DCIS should undergo individualized treatment.

An internal motor kinesin is associated with the Golgi apparatus and plays a role in trichome morphogenesis in Arabidopsis.

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.

Localization of two homologous Arabidopsis kinesin-related proteins in the phragmoplast.

During plant cytokinesis, kinesin-related motor proteins are believed to play critical roles in microtubule organization and vesicle transport in the phragmoplast. Previously, we reported that the motor AtPAKRP1 was associated with the plus end of phragmoplast microtubules in Arabidopsis thaliana [Lee Y-RJ, Liu B (2000) Curr Biol 10:797-800]. In this paper, we report a full-length cDNA from the same organism, which encodes a polypeptide 74% identical to AtPAKRP1. This AtPAKRP1-like protein--AtPAKRP1L--and AtPAKRP1 share similar domain structures along the polypeptides. Peptide antibodies were raised and purified to distinguish the two polypeptides in vitro and in vivo. When monospecific anti-AtPAKRP1 and anti-AtPAKRP1L antibodies were used in immunofluorescence, they both decorated the plus end of phragmoplast microtubules at all stages of phragmoplast development. Their localization patterns were indistinguishable from each other. By using bacterially expressed fusion proteins of motor-less versions of both polypeptides, it was revealed that AtPAKRP1 and AtPAKRP1L were able to interact with themselves and with each other. Using T-DNA insertional mutants, it was also demonstrated that AtPAKRP1 and AtPAKRP1L were not required for each other's localization. Our results therefore indicate that AtPAKRP1 and AtPAKRP1L are both expressed in the same cells, and likely have identical functions in the phragmoplast by forming either homodimers or heterodimers.