A family with segregation of an unbalanced translocation (7;13) (q36;q32) in three patients with severe mental retardation, microcephaly and dysmorphic features, detected by subtelomere FISH: genetic counselling and prenatal diagnosis.

We report a Sardinian family in which three members showed a mental-retardation-microcephaly-multiple malformations syndrome resulting from an unbalanced translocation (7;13)(q36;q32) which led to subtelomeric trisomy 7q36qter and partial monosomy 13q32qter. The unbalanced translocation was transmitted by alternate segregation from a female and a male carriers of the balanced translocation. The three patients had severe mental retardation, microcephaly and multiple minor facial and fingers anomalies. Neuroimages showed brain atrophy, associated in two patients with partial agenesis of the corpus callosum. FISH with chromosome 13 and 7 specific painting probes and subtelomere specific probes was instrumental for defining and characterizing the chromosomal translocation. Extensive genetic counseling and prenatal diagnosis has been offered to all the members of the family.

Genetic testing improves the diagnosis of adult type hypolactasia in the Mediterranean population of Sardinia.

OBJECTIVE: Recently, the C/T-13910 polymorphism on chromosome 2q21 in North-European populations has been found completely associated with lactase activity and its genetic typing proposed as first-stage screening test for adult hypolactasia. However, the C/T-13910 variant in some sub-Saharan African groups is not a predictor of lactase persistence. In this work, we wanted to verify if in the Mediterranean island of Sardinia, located in Southern Europe, the C/T-13910 polymorphism may be useful or not for the diagnosis of adult type hypolactasia. DESIGN: Validation study of a genetic testing for adult type hypolactasia in Sardinians. SETTING: Brotzu Hospital and Microcitemico Hospital, Cagliari, Italy. SUBJECTS: The sample consisted in 84 Sardinian individuals (63 women and 21 men; range 20-73 years) selected from a group of 832 patients. METHODS: Genetic testing was compared to an improved test obtained by a combination of different breath hydrogen tests and clinical assessment. RESULTS: We found that all 49 individuals with lactose malabsorption, demonstrated by a combination of different breath hydrogen tests and clinical assessment, carried the C/C-13910 genotype associated with lactase non-persistence, 23 individuals with lactose normal absorption carried the C/T-13910 genotype associated with lactase persistence and only one person with the above phenotype showed a discordant C/C-13910 genotype. The genetic testing showed very high sensitivity, specificity, positive and negative predictive values of 100, 95.8, 98 and 100%, respectively. CONCLUSIONS: Sardinians, unlike some ethnic groups in sub-Saharan Africa, show the same genetic association of hypolactasia with the C/T-13910 variant as other North-European populations. The genetic testing for the C/T-13910 variant may contribute to improving the diagnosis of adult type hypolactasia.

Frequency of the thiopurine S-methyltransferase alleles in the ancient genetic population isolate of Sardinia.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) is an enzyme involved in the normal metabolic inactivation of thiopurine drugs. Patients with intermediate or no TPMT activity are at risk of toxicity after receiving standard doses of thiopurine drugs and it was shown that inter-individual differences in response to these drugs is largely determined by genetic variation at the TPMT locus. OBJECTIVE: This study was designed to investigate in the Sardinian population the frequency distribution of four of the most common variants accounting for TPMT deficiency and to conduct comparative analyses with other populations in order to obtain insights into the main factors that have shaped diversity at the TPMT locus in Sardinia. METHODS: DNA was extracted in 259 Sardinians and the frequencies of allelic variants of TPMT were determined using polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: Among the 259 Sardinians genotyped, 6.95% were found to be heterozygous for one of four TPMT variants screened; for each variant the frequency estimate was 1.74%, 0.58%, 0.39% and 0.77% for TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C respectively. CONCLUSIONS: Although Sardinia does not show reduced diversity at the TPMT locus, the spectrum of TPMT allele frequencies affords evidence of remarkable influence of genetic drift and founder effects throughout its population history. In the broad context of the European TPMT diversity, the Sardinians come out as outliers, an observation consistent with previous genetic inferences that Sardinia has features of a genetic isolate.

The efficiency of in-situ hybridization on human chromosomes with alphoid DNAs is enhanced by previous digestion with AluI and TaqI.

Centromeric alphoid DNAs of human chromosomes 6, 9, 16 and Y were employed to obtain information on the molecular mechanism(s) determining cytological effects produced by digestion in situ with AluI and TaqI restriction enzymes, possibly related to the structure of the above-cited areas. The following cytological and biochemical experiments were carried out using the above-mentioned alphoid sequences as probes: (1) standard in-situ hybridization and in-situ hybridization after chromosome cleavage with AluI/TaqI, and (2) filter hybridization on the DNA fractions obtained from the material solubilized and that retained on the slides after digestion in situ with AluI/TaqI. Biochemical data show that cleavage of alphoid DNAs is not prevented by the peculiar organization of centromeric heterochromatin, but such cleavage is not necessarily followed by complete DNA solubilization. The analysis of alphoid sequence cleavage in naked genomic DNA as well as during digestion of fixed chromosomes shows that (1) AluI cuts more efficiently than TaqI, (2) DNA fragments as large as 3-5 kb can be solubilized, and (3) DNA fragments of the same size are found in both fractions of DNA, i.e. that retained on the chromosomes as well as that solubilized from chromosomes. Cytological data show that previous chromosome digestion, mostly with TaqI, increases the hybridization signal area, suggesting that this fact might be due to (1) chromatin reorganization produced by enzyme attack and/or (2) the presence of alphoid DNAs which might be restricted not only to the kinetochore area but also to para/peri-centromeric heterochromatin. Lastly, centromere DNA solubilization as a consequence of restriction enzyme cleavage seems to vary from chromosome to chromosome, thus suggesting that centromeric regions do not represent a homogeneous class of constitutive heterochromatin.

The effects of enzyme inactivation and incubation buffer on digestion in situ with restriction endonucleases.

Previous studies have shown that components of the incubation reaction other than the restriction endonucleases in an in situ restriction enzyme digest of chromosomes may induce G-like banding patterns. To determine whether factors other than DNA base composition play a role in determining restriction enzyme induced bands, we investigated the effect of reaction buffers alone or in the presence of heat inactivated enzymes. Our results show that enzymes such as AluI, RsaI and MspI become inactivated during 3-24 hr incubations at 37 C and that reaction buffers alone failed to produce G-like bands when inactive endonucleases were included.

Characterization of Dasypyrum villosum (L.) candargy chromosomal chromatin by means of in situ restriction endonucleases, fluorochromes, silver staining and C-banding.

The distribution of C-banded heterochromatin was determined in an inbred line of Dasypyrum villosum. Practically no difference in chromosome morphology or band distribution could be observed within the chromosomes of the same pair. Heterochromatin bands, revealed by Giemsa banding, were characterized by means of their differential reaction to fluorochromes, silver staining and in situ digestion with different restriction endonucleases. The results clearly indicate that in D. villosum two different classes of heterochromatin with different chromosomal local-ization exist: one is evidenced by both C-banding and DAPI staining and has mainly telomeric distribution, the other is evidenced only by C-banding and has mainly centromeric distribution.

The effects of aging on fixed DNA.

Agarose-gel electrophoretic analysis was carried out to study the effect(s) of aging on fixed naked DNA as well as on the DNA part of fixed chromosomes. Results show that aging of fixed DNA produces alterations in molecular size; the alteration is more effective in naked DNA than in the DNA part of fixed chromosomes; and the alteration occurs to a greater extent in air-dried DNA than in DNA aged in fixative. Since B-mercaptoethanol is capable of preventing DNA alterations induced by air-drying, oxidative processes are invoked for explaining these findings as well as the effect(s) of aging on fixed cytological preparations.

The DNA fragments produced by AluI and BstNI digestion of fixed mouse chromosomes.

AluI and BstNI restriction endonucleases were used to study cytological and biochemical effects on centromere DNA in fixed mouse chromosomes. These enzymes were employed, as it is known that AluI is incapable of attacking major satellite DNA, contrary to BstNI that is known to cut this DNA fraction into monomers of 234 bp. After digestion in situ, electrophoretic analysis was carried out to characterize the DNA purified (1) from the material remaining on the chromosomes and (2) from the material solubilized from chromosomes. The DNA was then transferred to a nylon filter and 32P-labelled major satellite DNA was used as a probe for hybridization experiments. Other preparations were simply stained with Giemsa after digestion in situ with AluI and BstNI. Our results show that although restriction endonuclease cleavage primarily depends on DNA base sequence, this factor is not always sufficient to explain nuclease-induced cytological effects. In fact, the structural organization of peculiar regions such as the centromeres of mouse chromosomes might affect cleavage efficiency when restriction enzyme digestion is performed in situ.

Factors affecting the digestion of restriction endonucleases in situ.

The influence of incubation buffers and glycerol on the enzyme activity of naked DNA of lambda phage and mouse, and of mouse chromosomal DNA was investigated. The results obtained varied in part from previously known data, but confirmed the importance of these factors in determining the patterns of in situ restriction enzyme digestion so far attributed exclusively to endonuclease activity.

Electron microscopy and biochemical analysis of mouse metaphase chromosomes after digestion with restriction endonucleases.

Electron microscopy (EM) of whole mounted mouse chromosomes, light microscopy (LM), and agarose gel electrophoresis of DNA were used to investigate the cytological effect on chromosomes of digestion with the restriction endonucleases (REs) AluI, HinfI, HaeIII and HpaII. Treatment with AluI produces C-banding as seen by LM, cuts DNA into small fragments, and reduces the density of centromeres and disperses the chromatin of the arms as determined by EM. Treatment with HinfI produces C-banding, cuts DNA into slightly larger fragments than does AluI and increases the density of centromeres and disperses the fibres in the chromosomal arms. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.

DNA alteration induced by ultraviolet light in human metaphase chromosomes substituted with 5'-bromodeoxy uridine: monitoring by monoclonal antibodies to double-stranded and single stranded DNA.

Fixed human metaphase chromosomes, whose DNA had been substituted with 5'-bromodeoxyuridine (BrdUrd) for two rounds of replication (TB/BB) or for one round in BrdUrd followed by another round in thymidine (TT/BT), were treated with ultraviolet light (UV), in the presence or in the absence of 33258 Hoechst, to produce sister chromatid differentiation (SCD). Giemsa staining was compared with staining with monoclonal antibodies to double-stranded or single-stranded DNA. We confirmed that UV acts by debrominating BrdUrd-stubstituted DNA but showed that debromination alone cannot explain all our findings. We postulated that UV-induced protein-protein cross-linking, occurring to a different extent in differently BrdUrd-substituted chromatids, may also be invoked in explaining our data. Lastly, the different behaviour of unifilarly substituted TB as opposed to BT chromatids in UV-treated chromosomes, allowed us to hypothesize that such chromatids may differ depending on whether or not newly synthesized DNA is formed on a BrdUrd-containing strand.

Nuclease activity in human metaphase chromosomes substituted with 5'-bromodeoxyuridine.

Human metaphase chromosomes, substituted with 5'-bromodeoxyuridine (BrdUrd) for one, two or three rounds of replication, were briefly pretreated with ultraviolet light (UV), in the presence of 33258 Hoechst, and subsequently digested with either exonuclease III or S1 nuclease. Pretreatment alone was not sufficient to induce sister chromatid differential staining (SCD), but allowed subsequent digestion with exonuclease III or S1. Such enzymes were found to induce SCD with ethidium bromide, as unifilarly BrdUrd-substituted chromatids (TB) were more resistant than bifilarly substituted chromatids (BB). Other experiments with DNase I or the AluI and HaeIII restriction endonucleases showed that only HaeIII was capable of inducing SCD by attacking BB more than TB chromatids preincubated with UV in the presence of Hoechst. SCD with exonuclease III/S1 nuclease seems to be due to (1) UV-induced DNA debromination occurring twice in BB as opposed to TB chromatids, and (2) alteration of chromatin protein structure occurring to a different extent in differently BrdUrd-substituted chromatids. Our findings with endonucleases, on the contrary, may depend on the capacity of enzymatic cleavage to cancel the different protein alterations induced differentially by UV in TB as opposed to BB chromatids.