Genetics of Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, also referred to as Müllerian agenesis, is the second most common cause of primary amenorrhea. It is characterized by congenital absence of the uterus, cervix, and the upper part of the vagina in otherwise phenotypically normal 46,XX females. MRKH syndrome has an incidence of about 1 in 4,500-5,000 newborn females and it is generally divided into two subtypes: MRKH type 1, in which only the upper vagina, cervix and the uterus are affected, and MRKH type 2, which is associated with additional malformations generally affecting the renal and skeletal systems, and also includes MURCS (MÜllerian Renal Cervical Somite) characterized by cervico-thoracic defects. MRKH syndrome is mainly sporadic; however, familial cases have been described indicating that, at least in a subset of patients, MRKH may be an inherited disorder. The syndrome appears to demonstrate an autosomal dominant inheritance pattern, with incomplete penetrance and variable expressivity. The etiology of MRKH syndrome is still largely unknown, probably because of its intrinsic heterogeneity. Several candidate causative genes have been investigated, but to date only WNT4 has been associated with MRKH with hyperandrogenism. This review summarizes and discusses the clinical features and details progress to date in understanding the genetics of MRKH syndrome.

Epigenetics of functional hypothalamic amenorrhea.

Functional hypothalamic amenorrhea (FHA) is a temporary infertility characterized by the suppression of the hypothalamic-pituitary-gonadal (HPG) axis, induced by the inhibition of the hypothalamic pulsatile secretion of the gonadotropin-releasing hormone (GnRH), in the presence of stressors, including eating disorders, excessive exercise, and psychological distress. Although the stressful factors that may lead to FHA are well-established, little is known about the inter-individual variability in response to stress and the consequent inhibition of the HPG axis. Not all women, indeed, manifest FHA in presence of stressful conditions. Recent studies highlighted a genetic contribution to FHA. Rare or polymorphic variants in genes that control the development and/or function of GnRH neurons may contribute, indeed, to the adaptability of the reproductive axis to stress factors. Also epigenetic changes have been associated with different pathways involved in the HPG axis and therefore, take part in FHA and confer a personal predisposition to anovulation consequent to a stressful event, or represent biological markers of response to stress. This review summarizes recent advances in the identification of the contribution of (epi)genetics to FHA and to long-term complications of functional amenorrhea, and reports insights into the involvement of additional genetic loci in FHA development on the bases of the clinical and molecular overlap with other gynecological and/or psychological conditions. Finally, we describe the promising application of induced pluripotent stem cells (iPSCs) as a new approach to investigate the molecular pathways involved in FHA.

ESX1 gene expression as a robust marker of residual spermatogenesis in azoospermic men.

BACKGROUND: It would be of value to identify ongoing spermatogenesis molecular markers which can predict successful sperm recovery in patients with non-obstructive azoospermia undergoing conventional or microsurgical testicular sperm extraction (TESE/microTESE). ESX1 is an X-linked homeobox gene expressed in testis, placenta, brain and lung in humans and specifically in pre- and post-meiotic germ cells of the testis in mice. METHODS: We investigated the sequence, expression (by RT-PCR) and epigenetic status (by promoter pyrosequencing) of ESX1 in testicular tissue samples, obtained from 81 azoospermic subjects in the context of surgical sperm extraction, to check a possible association between ESX1 alterations and impaired spermatogenesis, as determined by histological analysis. RESULTS: The ESX1 transcript was detected in 100% of cases diagnosed as obstructive azoospermia (33), hypospermatogenesis (18) and incomplete maturation arrest (MA) (2), and sperm recovery was also successful in 100% of these cases. ESX1 mRNA was also detected in 5 of 6 patients with incomplete Sertoli cell-only syndrome, in 4 of 6 subjects with complete MA but in only 3 of 16 cases of complete Sertoli cell-only syndrome (cSCOS), whereas sperm recovery was successful in 4 of 6, 2 of 6 and 5 of 16 of these patients, respectively. In cases of focal spermatogenesis, ESX1 expression and sperm retrieval were concordant in 14 of 19 (74%) cases subjected to TESE, but in only 3 of 11 (27%) men who underwent microTESE. With TESE, but not with microTESE, both samples originated from adjacent testicular areas. The pyrosequencing of the ESX1 CpG island revealed methylation levels that were significantly lower in ESX1 expressors when compared with non-expressors. CONCLUSIONS: ESX1 emerges as a potentially reliable spermatogenesis molecular marker, whose clinical value as a predictor of successful sperm retrieval warrants further studies.

[Early nutrition: the role of genetics and epigenetics]. / Nutrizione nelle prime epoche della vita: ruolo della genetica e dell' epigenetica.

Many adult diseases seem to be associated with early nutrition and the subsequent growth pattern. Epidemiological studies hypotized that babies with intrauterine and/or neonatal growth retardation may be at greater risk of metabolic syndrome later in life. According to the Barker's "thrifty phenotype hypotesis" early malnutrition, whereas inducing physiological compensation by the promotion of early survival, appears to confer greater susceptibility to adults diseases. Epigenetics, that is the interindividual variation in DNA methylation patterns and chromatin remodelling, provide a potential explanation for how environmental factors can modify the risk for development of many common diseases. Beginning from animal models, many studies concerning early nutrition, epigenetic modifications and genes expression have been carried out. Maternal undernutrition during pregnancy, especially in the peri-implantation period, not only causes a prolonged growth retardation but also modifies the programming of biochemical mechanisms related to endocrine-metabolic control. Human studies have demonstrated the role played by IGF-1 as indicator of nutritional status and fetal/postnatal growth retardation. It has been reported that alterations in IGF axis, which predispose to adults diseases, may be due to an alterated epigenetic regulation that can modify IGF expression. Despite the critical inter-relation between early nutrition, growth, development, and subsequent health, there are few data on the influence of early nutrition on the modifications of the epigenetic gear. Furthermore it is hoped for a bigger attention to the early nutrition to prevent the development of diseases later in life.

Chromosome 11 segmental paternal isodisomy in amniocytes from two fetuses with omphalocoele: new highlights on phenotype-genotype correlations in Beckwith-Wiedemann syndrome.

BACKGROUND: The phenotypic variability in Beckwith-Wiedemann syndrome (BWS) reflects the genetic heterogeneity of the mechanism which by default leads to the deregulation of genes located at 11p15.5. Genotype-phenotype correlation studies have demonstrated an association between omphalocoele and CDKN1C/p57 mutations or hypermethylation. Paternal uniparental disomy 11 (pUPD11) has been described only in the mosaic condition with both uniparental and biparental cell lines, and no association with omphalocoele has been pointed out. METHODS: Two cases are presented here, in which a paternal segmental UPD11 was detected by molecular investigation of amniotic fluid cell cultures after the presence of apparently isolated omphalocoele was revealed in the fetuses by ultrasound scan. Further studies were performed on additional autoptic feto-placental tissues to characterise the distribution of the uniparental cell line and to unmask any biparental lineage in order to document in more detail the as yet unreported association between omphalocoele and pUPD11. RESULTS: Results on the UPD distribution profile showed that the abdominal organs have a predominant uniparental constitution. This condition could mimic the effect of CDKN1C/p57 inactivation, causing the omphalocoele. CONCLUSION: New genotype-phenotype correlations emerge from the investigated cases, suggesting that molecular analysis be extended to all cases with fetal omphalocoele in order to establish the incidence of pUPD11 in complete BWS and in monosymptomatic/mild forms.

Characterization of multi-locus imprinting disturbances and underlying genetic defects in patients with chromosome 11p15.5 related imprinting disorders.

The identification of multilocus imprinting disturbances (MLID) appears fundamental to uncover molecular pathways underlying imprinting disorders (IDs) and to complete clinical diagnosis of patients. However, MLID genetic associated mechanisms remain largely unknown. To characterize MLID in Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, we profiled by MassARRAY the methylation of 12 imprinted differentially methylated regions (iDMRs) in 21 BWS and 7 SRS patients with chromosome 11p15.5 epimutations. MLID was identified in 50% of BWS and 29% of SRS patients as a maternal hypomethylation syndrome. By next-generation sequencing, we searched for putative MLID-causative mutations in genes involved in methylation establishment/maintenance and found two novel missense mutations possibly causative of MLID: one in NLRP2, affecting ADP binding and protein activity, and one in ZFP42, likely leading to loss of DNA binding specificity. Both variants were paternally inherited. In silico protein modelling allowed to define the functional effect of these mutations. We found that MLID is very frequent in BWS/SRS. In addition, since MLID-BWS patients in our cohort show a peculiar pattern of BWS-associated clinical signs, MLID test could be important for a comprehensive clinical assessment. Finally, we highlighted the possible involvement of ZFP42 variants in MLID development and confirmed NLRP2 as causative locus in BWS-MLID.

Microsatellite alterations in bronchial and sputum specimens of lung cancer patients.

Early diagnosis of lung cancer based on conventional screening procedures has been unable thus far to decrease lung cancer mortality. We explored the possibility of using microsatellite instability in the detection and screening of early phases of lung carcinogenesis. We studied tumor, histopathologically normal bronchial mucosa, and cytological specimens of 51 lung cancer patients for the presence of clonal variations at microsatellite polymorphisms. Microsatellite alterations were found in tumor, normal bronchial mucosa and cytological specimens of 25 of 51 (49%) of the patients. The detection of microsatellite alterations in histopathologically normal bronchial specimens and cytological clinical samples with minimal atypia suggests a possible application of this genetic marker in early diagnosis of precancerous lesions and lung cancer.

Deletions of 17p and p53 mutations in preneoplastic lesions of the lung.

Cytogenetic and p53 mutation analysis in two cases of severe dysplasia of the bronchial epithelium in lung cancer patients and p53 immunostaining in a third one are reported. The finding of both chromosomal deletions of 17p and p53 mutation indicates that these changes may take place early in the process of lung carcinogenesis.

Cytogenetic abnormalities and overexpression of receptors for growth factors in normal bronchial epithelium and tumor samples of lung cancer patients.

A cytogenetic analysis was performed on direct preparations and short-term cell cultures of lung tumor and normal bronchial epithelium of 19 patients carrying either a first or a second primary lung cancer. In 9 tumors (6 squamous cell carcinomas, 1 adenocarcinoma, 1 mucoepidermoid carcinoma, and 1 small cell lung carcinoma) successfully analyzed, pseudodiploid and hyperdiploid karyotypes were observed with a heterogeneous pattern of chromosome abnormalities but with a consistent involvement (5 cases) of the short or the long arm of chromosome 3. The normal bronchial epithelial cells had a normal karyotype in 11 patients, whereas in 6 patients clonal and nonclonal chromosomal abnormalities were observed. Involvement of chromosome 7 was present in 4 cases. In addition, overexpression of the growth factor receptors, epidermal growth factor receptor and HER-2/neu, was found in 9 of 18 tumors and in 6 of 13 bronchial epithelium samples. These findings suggest that early genetic lesions could be present in the normal bronchial epithelial cells that are the target of further complex and multiple genetic changes occurring during the pathogenesis of lung cancer.

Genetic evidence for an independent origin of multiple preneoplastic and neoplastic lung lesions.

Patients with a primary cancer in the lung or in the upper aerodigestive tract have an increased risk of developing synchronous or metachronous second primary lung tumors. This phenomenon has been related to the chronic exposure of the bronchial tree to carcinogens through a so-called "field cancerization" process. This study was designed to investigate at the somatic level the genetic basis of the field cancerization effect in patients having multiple simultaneous neoplastic and preneoplastic lesions of the lung. The pattern of specific genetic changes occurring with high frequency and in early stages of lung carcinogenesis including p53 mutations, deletions of chromosome 3p, and K-ras mutations, was investigated by immunocytochemical, cytogenetic, and molecular approaches in 11 synchronous lesions of five patients with multiple lung cancers. Different genetic lesions were observed in all of the pathological specimens analyzed from each patient. The pattern of these changes was different both in topographically distant or adjacent lesions and in tumors with the same histopathological diagnosis supporting their independent origin. The present data provide further evidence of the clinical relevance of the field cancerization process, and support the use of genetic markers in the differential diagnosis of recurrence or metastasis versus second primaries of the lung.

Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise.

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.

Human TRK proto-oncogene maps to chromosome 1q32-q41.

The chromosomal localization of TRK, a gene coding for a putative receptor molecule with an associated tyrosine kinase activity that we have found activated in 25% of patients with papillary thyroid carcinoma, was determined by Southern blot analysis of a panel of human-rodent somatic cells using a cDNA clone containing the entire human TRK proto-oncogene (Martin-Zanca et al., 1986). The TRK gene was assigned to chromosome 1. One hybrid that had retained only the short arm of the human chromosome 1 was negative. Subsequently, in situ hybridization of the same probe to human metaphase chromosomes localized the TRK gene to 1q32-q41.

The human tropomyosin gene involved in the generation of the TRK oncogene maps to chromosome 1q31.

The chromosomal localization of hTMnm, a gene coding for a cytoskeletal tropomyosin non-muscle isoform involved in the activation of the TRK proto-oncogene in various human tumors, was determined by Southern blot analysis of a panel of human-rodent somatic cell hybrids. Using as a probe an Alu-free intronic fragment related to the tropomyosin sequence fused to the TRK tyrosine kinase domain, the hTMnm gene was assigned to the long arm of chromosome 1. Subsequently, in situ hybridization of the same probe to human metaphase chromosomes localized the hTMnm gene to 1q31. Since we have recently assigned the TRK locus to chromosome 1q32-q41, the generation of the hybrid transforming sequence tropomyosin-TRK may be due to an intrachromosomal rearrangement of the long arm of chromosome 1.

Refined localization to contiguous regions on chromosome 10q of the two genes (H4 and RET) that form the oncogenic sequence PTC.

In this report we confirm the localization of the human RET proto-oncogene to chromosome 10q11.2, both by Southern blot analysis of a panel of human-rodent somatic cell hybrids and by in situ hybridization on human metaphase chromosomes. Previously, we had assigned to the same chromosome region the gene termed H4. In about 25% of papillary thyroid carcinomas, this gene was shown to rearrange with RET to give rise to the transforming sequence PTC. The analysis of different cell hybrids containing subfragments of chromosome 10, in conjunction with pulse field gel electrophoresis, established that H4 is mapped distally to RET at a distance not less than 280 kb. These findings suggest that intrachromosomal rearrangements are responsible for PTC activation in papillary thyroid carcinomas.

The oncogene associated with human papillary thyroid carcinoma (PTC) is assigned to chromosome 10 q11-q12 in the same region as multiple endocrine neoplasia type 2A (MEN2A).

In this report we assigned to chromosome 10q the human oncogene PTC frequently associated with the papillary type of thyroid carcinoma. Using an informative panel of human-mouse somatic cell hybrids and 'in situ' hybridization to human metaphase chromosomes, we localized the PTC gene at bands q11-q12 of chromosome 10. These bands belong to one of the two regions on chromosome 10 linked to the cancer syndrome multiple endocrine neoplasia type 2A (MEN2A). Therefore, it is suggested that genes clustered in certain regions of chromosome 10 could be involved in the developmental regulation of the thyroid gland.

Fetal and placental chromosomal mosaicism revealed by QF-PCR in severe IUGR pregnancies.

A number of genetic and environmental factors are taken into account as responsible for intrauterine growth restriction (IUGR); nevertheless, the relevance of genetic alteration in IUGR aetiology remains to be determined. The aim of this study was to investigate using a combined cytogenetic-molecular approach, improved by a new application of QF-PCR method, the presence of mosaic chromosomal changes in fetal/placental samples from 12 pregnancies with unexplained severe IUGR. This multiple approach allowed us to reveal and quantify subtle chromosomal mosaicisms with less than 5% of trisomic cells even in cases in which cytogenetic and FISH analyses failed to reveal them. These are three pregnancies with a mosaic trisomy for chromosomes 7, 2 and 14; the former case presented matUPD7 and was previously described in this journal (Placenta 22 (2001) 813) in association with pre- and postnatal growth restriction. It is intriguing that chromosomes 7, 2 and 14 are known or suspected to harbour imprinted genes, so that an unbalanced gene dosage in a subset of cells during embryonic development could lead to an early impairment of placental function. Our findings indicate that extensive molecular and cytogenetic studies of IUGR fetal and placental tissues are necessary to reveal at least part of the heterogeneous genetic lesions implicated in IUGR phenotypes.

The amphiphilic character of glycogenin.

This study describes for the first time the amphiphilicity of the protein moiety of proteoglycogen. Glycogenin but not proteoglycogen associates to phospholipid vesicles and forms by itself stable Gibbs and Langmuir monolayers at the air-buffer interface. The adsorption free energy (-6.7 kcal/mol) and the glycogenin collapse pressure (47 mN/m) are indicative of its high surface activity which can thermodynamically drive and retain the protein at the membrane interface to a maximum equilibrium adsorption surface pressure of 21 mN/m. The marked surface activity of glycogenin is further enhanced by its thermodynamically favorable penetration into zwitterionic and anionic phospholipids with a high cut-off surface pressure point above 30 mN/m. The strong association to phospholipid vesicles and the marked surface activity of glycogenin correspond to a high amphiphilic character which supports its spontaneous association to membrane interfaces, in which the de novo biosynthesis of glycogen was proposed to initiate.

Post-zygotic origin of complete maternal chromosome 7 isodisomy and consequent loss of placental PEG1/MEST expression.

Maternal UPD of chromosome 7 is associated with pre- and postnatal growth retardation (IUGR, PNGR) and Silver-Russell syndrome (SRS [MIM 180860]). We report a case of IUGR in a newborn with SRS stigmata. Using combined haplotyping and cytogenetic-FISH studies we characterized the lymphocytes, umbilical cord and four placental cotyledons. The results are consistent with complete maternal isodisomy 7 and trisomy 7 mosaicism of post-zygotic origin. The trisomic cell line was prevalent in trophoblast cells from two placental cotyledons. Trisomy 7 of post-zygotic origin is a frequent finding, but maternal isodisomy 7, due to trisomic rescue has never been reported. PEG1/MEST expression was evaluated on placenta cDNA and a specific transcript was revealed only in the cotyledons with a high percentage of trisomic cells and the presence of the paternal chromosome 7 contribution, but not in the placental biopsies with maternal isodisomy 7. The histological features of the four placental fragments revealed that isodisomy 7 correlates with a pattern of cotyledonary hyper-ramification due to an increase of the branching angiogenesis, which could be the result of a defect of angiogenesis caused by the absence of PEG1 product. The severe hypo-ramification of the two cotyledons, showing trisomy 7 mosaicism, may be due to the triplicate dosage of genes on chromosome 7. The delayed fetal growth could be the phenotypic effect of the imbalance between imprinted and non-imprinted genes on chromosome 7 in the fetus or the result of abnormal placental function during pregnancy.

Fetal growth restriction: a workshop report.

Intrauterine growth restriction (IUGR) is associated with significantly increased perinatal morbidity and mortality as well as cardiovascular disease and glucose intolerance in adult life. A number of disorders from genetic to metabolic, vascular, coagulative, autoimmune, as well as infectious, can influence fetal growth by damaging the placenta, leading to IUGR as a result of many possible fetal, placental and maternal disorders. Strict definitions of IUGR and of its severity are needed in order to eventually distinguish among different phenotypes, such as gestational age at onset, degree of growth restriction and presence of hypoxia. This report explores and reviews some of the most recent developments in both clinical and basic research on intrauterine growth restriction, by seeking mechanisms that involve genetic factors, utero-placental nutrient availability and vascular growth factors. New exciting findings on the genomic imprinting defects potentially associated with IUGR, and the placental anomalies associated with the decreased nutrient transport are summarized. Moreover, recent data on angiogenic growth factors as well as new information arising from application of gene chip technologies are discussed.