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M P Lombardi

Publications and source records attributed to M P Lombardi.

10 recordsLinked to original sources
PubMed2006

Mutations in the testis-specific NALP14 gene in men suffering from spermatogenic failure.

BACKGROUND: Because of the common use of ICSI and the potential genetic aetiology of spermatogenic failure, concern has been raised about transmitting genetic disorders to ICSI offspring. However, to date, in only approximately 15% of all cases of spermatogenic failure, an underlying genetic cause can be identified. We have previously established an association between spermatogenic failure and chromosomal region 11p15. In this study, we set out to explore whether NALP14, a gene recently mapped to 11p15, has a function in spermatogenesis and whether mutations in NALP14 can cause spermatogenic failure. METHODS: We applied two different multiple tissue northern (MTN) blots to determine tissue specificity of NALP14 and performed immunohistochemistry on human testis with anti-NALP14 antiserum. To determine imprinting status of NALP14, we tested the expression pattern of two single-nucleotide polymorphisms (SNPs) in human testis. Finally, we performed a mutation screen of the NALP14 gene in 157 men with azoospermia or severe oligozoospermia by direct sequencing; 158 normospermic men served as controls. RESULTS: NALP14 was, as are the three other genes in 11p15, exclusively expressed in testis. Within the testis, the NALP14 protein was mainly expressed in A dark spermatogonia, mid and late spermatocytes and spermatids. The mutation screen revealed five mutations that occurred only in the patient group. One of these unique mutations introduced an early stop codon in the NALP14 sequence, predicted to result in a severely truncated protein. CONCLUSION: Our data suggest that NALP14 has a function in spermatogenesis and that mutations in this gene might cause spermatogenic failure.

Amino Acid Sequence↗
PubMed2006

Molecular genetic testing for familial hypercholesterolemia in the Netherlands: a stepwise screening strategy enhances the mutation detection rate.

Familial hypercholesterolemia (FH) has been identified as a major risk factor for coronary vascular disease and is associated with mutations in the low-density liporotein receptor (LDLR) and apolipoprotein B (APOB) gene. The molecular basis of FH in the Dutch population is well understood. Approximately 160 different LDLR and APOB gene defects have been identified with a panel of 9 LDLR gene and 1 APOB gene frequently occurring mutations accounting for approximately 30% of all clinically diagnosed FH cases. As molecular diagnosis of FH is becoming increasingly widely applied, a variety of mutation detection rates is reported, ranging from as low as 30% and up to 80%. This variability appears to depend on the clinical criteria applied to identify patients with FH and on the strategies and methodologies used for mutation screening. In this study we describe the application of a stepwise screening approach, combining different methodologies, to detect mutations of the LDLR gene and APOB gene in 1465 patients with FH. A mutation was found in approximately 44% of the patients, which demonstrates that this is an effective strategy for the molecular diagnosis of FH.

Adolescent↗
PubMed2005

Mutations in the chromosome pairing gene FKBP6 are not a common cause of non-obstructive azoospermia.

Although it is generally thought that spermatogenic failure has a genetic background, to date only a limited percentage of men with non-obstructive azoospermia (NOA) are diagnosed with a genetic defect. The only common and well-established genetic causes of NOA in humans are numerical and structural chromosomal abnormalities and Y-chromosome deletions. In addition, some infrequent mutations have been identified in the ubiquitin-specific protease 9, Y-linked (USP9Y) and the synaptonemal complex protein 3 (SYCP3) gene that cause azoospermia. FK506-binding protein 6 (Fkbp6) is a newly discovered component of the synaptonemal complex (SC), which is essential for proper chromosome pairing and meiotic division. A null mutation of the Fkbp6 gene causes azoospermia in mice as well as in rats. We tested the hypothesis whether mutations in this gene can also cause azoospermia in humans. We performed a mutation screen in 51 men with NOA through direct sequencing methods. No homozygous mutations were identified. Two heterozygous mutations (T173T and R183C) were identified, which are likely to disrupt FKBP6 protein function. However, both mutations were also found in a group of 218 normospermic controls indicating that one FKBP6 allele appears to be sufficient for normal spermatogenesis. In conclusion, our results suggest that genetic defects in FKBP6 can be excluded as a common cause of azoospermia in humans.

Azoospermia↗
PubMed2004

Heterogeneous nuclear ribonucleoprotein G-T (HNRNP G-T) mutations in men with impaired spermatogenesis.

The genetic cause of male subfertility due to impaired spermatogenesis is unknown in the majority of cases, but the general assumption is that it is a complex disorder. The aim of this study was to determine whether mutations occur in the HNRNP G-T gene in men with idiopathic impaired spermatogenesis. The heterogeneous nuclear ribonucleoprotein G-T (HNRNP G-T) gene is located in chromosomal region 11p15 that has been shown to be associated with impaired spermatogenesis. It is a member of the hnRNP gene family and is predominantly expressed in pachytene spermatocytes and round spermatids, where it is thought to affect splicing and signal transduction. We identified eight single nucleotide variants in our patient group of 153 subfertile men by sequencing the HNRNP G-T gene. Two of the mutations, R100H and G388del, did not occur in a control group of 143 normozoospermic men. The R100H mutation causes loss of a conserved arginine, thereby affecting a putative site of methylation possibly required for RNA-binding. Interestingly, this mutation was inherited from the mother. The G388del mutation causes loss of one non-conserved glycine located in a glycine stretch at the end of the protein that is not a known functional motif or domain. Our data show that HNRNP G -T mutations are not a frequent cause of impaired spermatogenesis. Nevertheless, the R100H mutation detected suggests that in some men mutations in the HNRNP G-T gene can cause impaired spermatogenesis.

Amino Acid Sequence↗
PubMed2000

Molecular genetic testing for familial hypercholesterolemia: spectrum of LDL receptor gene mutations in The Netherlands.

Mutations in the LDL receptor are responsible for familial hypercholesterolemia (FH). At present, more than 600 mutations of the LDL receptor gene are known to underlie FH. However, the array of mutations varies considerably in different populations. Therefore, the delineation of essentially all LDL receptor gene mutations in a population represents a prerequisite for the implementation of nation-wide genetic testing for FH. In this study, the frequency and geographical distribution of 13 known mutations were evaluated in a cohort of 1223 FH patients. We identified 358 mutation carriers, representing 29% of the FH cohort. Four mutations (N543H-2393de19, 1359--1G-->A, 313 + 1 G-->A and W23X) occurred with a relatively high frequency, accounting for 22.4% of the entire study cohort. Two of these common FH mutations (N543H-2393de19 and 1359 - 1G-->A) showed a preferential geographic distribution. Second, to further expand the array of LDL receptor gene mutations, we conducted mutation analysis by denaturing gradient gel electrophoresis (DGGE) in 141 children with definite FH. A mutation was identified in 111 patients, involving 16 new single base substitutions and four small deletions and insertions, which brings the number of different FH-causing mutations in our country up to 61. Our data indicate that an estimate of the prevalence of specific mutations, as well as the compilation of a database of all FH-causing mutations in a given country, can facilitate selection of the most appropriate molecular diagnostic approach.

Child↗
PubMed1998

Similar response to simvastatin in patients heterozygous for familial hypercholesterolemia with mRNA negative and mRNA positive mutations.

In patients heterozygous for familial hypercholesterolemia, the low-density lipoprotein (LDL) cholesterol lowering effect of beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors may depend on the nature of the mutation in the LDL receptor gene. To test this hypothesis, we compared the response to simvastatin, 20 mg daily for 9 weeks, between heterozygous carriers of functionally different classes of mutations, i.e. mRNA negative or mRNA positive mutations. Out of 116 consecutive, unrelated patients with familial hypercholesterolemia, 27 patients were selected for molecular analyses: 14 patients with mRNA negative and 13 with mRNA positive mutations. Before simvastatin treatment, patients with mRNA negative mutations had higher levels of LDL cholesterol, lower levels of high-density lipoprotein (HDL) cholesterol and significantly more often tendon xanthomas, compared to patients with mRNA positive mutations. Simvastatin reduced the mean fasting LDL cholesterol levels to a similar percentage in the mRNA negative and mRNA positive patients (37, 36%, respectively, 95% CI of difference--8 to 5%, P = 0.2). This effect was similar to the 37% decrease observed in our total series of patients with familial hypercholesterolemia (n = 116). The increase in mean concentration of HDL cholesterol was greater in the mRNA negative group than in the mRNA positive group (16, 0%, respectively, 95%, CI of difference 8-25%, P = 0.002) independent of the response of total triglycerides to simvastatin. The percentage LDL cholesterol lowering response varied among multiple carriers of the same mutation, even in the case of mRNA negative mutations. We conclude that the percentage LDL lowering response to simvastatin treatment was similar in patients with mRNA negative and mRNA positive mutations. Moreover, variation of this response within multiple carriers of the same mutation suggests an influence of additional factors.

Anticholesteremic Agents↗
PubMed1997

Mutational and genetic origin of LDL receptor gene mutations detected in both Belgian and Dutch familial hypercholesterolemics.

DNA samples from 100 unrelated Belgian patients with familial hypercholesterolemia (FH) were screened for the presence of specific low-density lipoprotein receptor (LDLR) gene mutations, previously shown to be prevalent in related populations. Two point mutations, viz., P664L and a G to A splicing defect at position 1359-1, were detected in single Flemish-speaking families. A long-distance polymerase chain reaction (PCR) assay, used to screen for the 4-kb and 2.5-kb deletions previously identified by Southern blot analyses in different parts of The Netherlands, revealed a 3-kb deletion in two Belgian patients. Comparison of PCR product length showed that both Dutch deletions of exons 7-8 are identical to that found in Belgians, but different from the 2.5-kb deletion previously described in South Africans of mixed ancestry. The Belgian patients probably share a common ancestor, for each mutation identified, with FH patients from The Netherlands, since all three mutations were associated with the same LDLR gene haplotype as described for the Dutch population. Analysis of the deletion junctions demonstrated the role of a 31-bp repetitive sequence in the generation of large rearrangements involving exons 7 and 8 of the LDLR gene. The finding that only 4 out of 100 analyzed Belgian hypercholesterolemics carry a known LDLR mutation that is prevalent in related populations suggests that the Belgian FH population has its own spectrum of mutations.

Adolescent↗
PubMed1996

Detection of cellular heterogeneity by DNA ploidy, 17 chromosome, and p53 gene in primary carcinoma and metastasis in a case of ovarian cancer.

An unusual case of a patient with ovarian carcinoma carrying the p53 point mutation in both metastases (omentum and lymph node), but not in the primary tumor, is described. The presence of a p53 single mutation (G:A) at the second base of codon 248 was examined by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) analysis. This case was examined also by fluorescent in situ hybrization (FISH) analysis and flow cytometry (FCM) to obtain further information at the single cell level and to detect heterogeneity within a population of cells. FCM analysis evidenced the same multiple aneuploid cell subpopulations in primary and in metastatic samples showing the presence of a cellular heterogeneity. FISH analysis showed a disomic condition for the 17 chromosome in the primary and in one metastasis, while in the other metastasis a monosomic together with a disomic subpopulation was revealed. Our results confirm the independent clonal evolution of the metastasis. The late mutation event observed only in metastatic specimens suggests the hypothesis that in the primary tumor the wild-type gene either does not perform its control role for unknown genetic structural events or the p53 gene in this case does not play a critical role in carcinogenesis.

Base Sequence↗
PubMed1995

Antimitotic and antiviral activities of Kelletinin A in HTLV-1 infected MT2 cells.

Kelletinin A [ribityl-pentakis (p-hydroxybenzoate)] (KA), a natural compound isolated from the marine gastropod Buccinulum corneum, showed antiviral activity on the human T-cell leukemia virus type-1 (HTLV-1) and antimitotic activity on HTLV-1-infected MT2 cells. KA inhibited cellular DNA and RNA synthesis, without influencing protein synthesis, and interfered with viral transcription by reducing the levels of high molecular weight transcripts. Finally, the compound inhibited HTLV-1 reverse transcriptase in vitro.

Antineoplastic Agents↗