Comprehensive clinical and molecular analysis of 12 families with type 1 recessive cutis laxa.

Autosomal recessive cutis laxa type I (ARCL type I) is characterized by generalized cutis laxa with pulmonary emphysema and/or vascular complications. Rarely, mutations can be identified in FBLN4 or FBLN5. Recently, LTBP4 mutations have been implicated in a similar phenotype. Studying FBLN4, FBLN5, and LTBP4 in 12 families with ARCL type I, we found bi-allelic FBLN5 mutations in two probands, whereas nine probands harbored biallelic mutations in LTBP4. FBLN5 and LTBP4 mutations cause a very similar phenotype associated with severe pulmonary emphysema, in the absence of vascular tortuosity or aneurysms. Gastrointestinal and genitourinary tract involvement seems to be more severe in patients with LTBP4 mutations. Functional studies showed that most premature termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This correlated with increased transforming growth factor-beta (TGFß) activity. However, one mutation, c.4127dupC, escaped nonsense-mediated decay. The corresponding mutant protein (p.Arg1377Alafs(*) 27) showed reduced colocalization with fibronectin, leading to an abnormal morphology of microfibrils in fibroblast cultures, while retaining normal TGFß activity. We conclude that LTBP4 mutations cause disease through both loss of function and gain of function mechanisms.

Meiotic segregation of a t(7;8)(q11.21;cen) translocation in two carrier brothers.

OBJECTIVE: To determine the meiotic segregation of a t(7;8)(q11.21;cen) translocation in two carrier brothers. DESIGN: Analysis of sperm nuclei by fluorescence in situ hybridization (FISH). SETTING: Franche-Comté University Fertility Center in Besançon, France. PATIENT(S): Two oligospermic brothers with a (7;8) translocation. INTERVENTION(S): Dual FISH for chromosomes 7 and 8, and multicolor FISH for chromosomes X, Y, 15, and 18 were performed. MAIN OUTCOME MEASURE(S): Meiotic segregation differences between both brothers and controls. RESULT(S): In dual FISH 7-8, a total of 34527 (older brother) and 10023 (younger brother) spermatozoa were analyzed. The frequencies of alternate, adjacent 1, adjacent 2, and 3:1 segregations and diploidies were 56.7%, 25.1%, 11.1%, 7.06%, and 0.04%, respectively, in the older brother and 62.84%, 17.61%, 12.8%, 6.47%, and 0.28% in the younger. Also, the disomy rates of some chromosomes not implicated in the translocation would suggest a possible interchromosomal effect. CONCLUSION(S): The similar segregation profiles for the same translocation, compared with those very divergent profiles of the other cases of studied translocations that were published in the literature, confirm that the risks of meiotic imbalances vary primarily according to the characteristics of the chromosomes involved in the rearrangement and the breakpoint position.

The semen quality of 1158 men with testicular cancer at the time of cryopreservation: results of the French National CECOS Network.

Testicular cancer is the most common cancer in young men. Several studies have reported an alteration in semen quality in nonseminoma tumors, but this result has not been confirmed in all of the published data. We performed a retrospective study in a population of 1158 men with testicular cancer who banked sperm between 1999 and 2003 in 11 French Centre d'Etude et de Conservation des Oeufs et du Sperme humain laboratories. Our study evaluated prefreeze and postthaw sperm parameters according to patient medical history, tumor histological type, and disease stage. Pure seminomas were found in 48% of our population. Testicular cancer was generally diagnosed at stage I. In cases of a history of unilateral cryptorchidism, testicular cancer occurred preferentially in the maldescended testis. Semen samples were preferentially collected after orchiectomy. The sperm concentration and total sperm number were significantly lower before orchiectomy in seminomas compared with nonseminoma tumors (P < .001). After orchiectomy, these parameters decreased for nonseminoma tumors and did not vary for seminomas. Semen parameters were more severely impaired for stage III tumors, and when patients had a history of cryptorchidism or when they were less than 20 years of age. Azoospermia was more frequently observed before than after orchiectomy. In this study, we determined that sperm cryobanking should preferentially be performed before orchiectomy and that testicular sperm extraction concurrent with orchiectomy should be used in severe spermatogenesis impairment. Our study highlights that seminomas alter sperm production more significantly than nonseminoma tumors and seem to preferentially impair spermatogenesis in tumor-bearing testes.

Child with Beckwith-Wiedemann syndrome born after assisted reproductive techniques to an human immunodeficiency virus serodiscordant couple.

OBJECTIVE: To report a child with Beckwith-Wiedemann syndrome (BWS) born after assisted reproductive technology (ART) to an HIV serodiscordant couple. DESIGN: Case report. SETTING: Academic medical center. PATIENT(S): A child with BWS born after ART to an HIV serodiscordant couple. INTERVENTION(S): Assisted reproductive techniques. MAIN OUTCOME MEASURE(S): ART and HIV. RESULT(S): Since 2003, it has been suggested that there is an association between ART and BWS, which is a congenital overgrowth syndrome characterized by macrosomia, macroglossia, visceromegaly, umbilical and abdominal wall abnormalities, and an increased risk of developing embryonal tumors in childhood. It is a multigenic disorder resulting from genetic or epigenetic alterations of genes on chromosome 11p15. Methylation errors account for 50%-60% of sporadic cases and almost 100% of cases born after ART. We report the birth of a child diagnosed with BWS arising from an ART cycle to an HIV serodiscordant couple with no history of infertility. This case cannot constitute conclusive evidence but it raises various questions. CONCLUSION(S): Assisted reproductive technology seems to be in the causal pathway but a male/female factor or an iatrogenic factor is also possible.

Notable contribution of large CFTR gene rearrangements to the diagnosis of cystic fibrosis in fetuses with bowel anomalies.

Grade III fetal bowel hyperechogenicity and/or loop dilatation observed at the second trimester of pregnancy can be due to several disease conditions, including cystic fibrosis (CF). Screening for frequent CF mutations is performed as a first step and, in certain situations, such as when a frequent CF mutation is found in the fetus, the increased risk of CF justifies an in-depth study of the second allele. To determine the contribution of large CFTR gene rearrangements in such cases, detected using a semiquantitative fluorescent multiplex PCR (QFM-PCR) assay, we collated data on 669 referrals related to suspicion of CF in fetuses from 1998 to 2009. Deletions were found in 5/70 cases in which QFM-PCR was applied, dele19, dele22_23, dele2_6b, dele14b_15 and dele6a_6b, of which the last three remain undescribed. In 3/5 cases, hyperechogenicity was associated with dilatation and/or gallbladder anomalies. Of the total cases of CF recognized in the subgroup of first-hand referrals, deletions represent 16.7% of CF alleles. Our study thus strengthens the need to consider large CFTR gene rearrangements in the diagnosis strategy of fetal bowel anomalies, in particular in the presence of multiple anomalies.

Prevalence and spectrum of mutations in a cohort of 192 unrelated patients with hypertrophic cardiomyopathy.

Hypertrophic Cardiomyopathy (HCM), a common and clinically heterogeneous disease characterized by unexplained ventricular myocardial hypertrophy and a high risk of sudden cardiac death, is mostly caused by mutations in sarcomeric genes but modifiers genes may also modulate the phenotypic expression of HCM mutations. The aim of the current study was to report the frequency of single and multiple gene mutations in a large French cohort of HCM patients and to evaluate the influence of polymorphisms previously suggested to be potential disease modifiers in this myocardial pathology. We report the molecular screening of 192 unrelated HCM patients using denaturing high-performance liquid chromatography/sequencing analysis of the MYBPC3, MYH7, TNNT2 and TNNI3 genes. Genotyping of 6 gene polymorphisms previously reported as putative HCM modifiers (5 RAAS polymorphisms and TNF-α -308 G/A) was also performed. Seventy-five mutations were identified in 92 index patients (48%); 32 were novel. MYBPC3 mutations (25%) represent the most prevalent cause of inherited HCM whereas MYH7 mutations (12%) rank second in the pathogenesis. The onset age was older in patients carrying MYBPC3 mutations than in those with MYH7 mutations. The MYBPC3 IVS20-2A>G splice mutation was identified in 7% of our HCM population. Multiple gene mutations were identified in 9 probands (5%), highlighting the importance of screening other HCM-causing genes even after a first mutation has been identified, particularly in young patients with a severe phenotype. No single or cumulative genetic modifier effect could be evidenced in this HCM cohort.

Quantitative PCR technique for the identification of microrearrangements of the AZFc region.

PURPOSE: The AZFc region spans about 3.5 Mb and contains many amplicons causing recombination events. Several papers have reported the occurrence of AZFc partial deletions resulting from non allelic homologous recombination (NAHR) ("gr-gr", "b1-b3" or "b2-b3" deletions), particularly in infertile patients. DAZ genes are present in 4 copies and rearrangements involve a modification of the number of DAZ genes. METHODS: In addition to STS plus/minus PCR, we developed a quantitative technique using real time PCR (Q-PCR) to determine the number of DAZ genes. Fourteen DNA controls were selected to validate the use of Q-PCR to detect AZFc microrearrangements, and sperm DNA samples from 30 fertile men were studied. RESULTS: Rearrangements of 14 controls were well identified with Q-PCR, and 2 AZFc partial deletions were detected in fertile men (1 "gr-gr" and 1 "b2-b3"). CONCLUSION: Q-PCR represents a well-adapted method to detect microrearrangements of the Y-chromosome, complementary to STS analysis.

Use of trypan blue staining to assess the quality of ovarian cryopreservation.

OBJECTIVE: To check the efficiency of the cryopreservation procedure by using trypan blue staining of ovarian test fragments to assess the quality of frozen-thawed ovarian tissue. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with polycystic ovary syndrome undergoing laparoscopic ovarian drilling. INTERVENTION(S): Ovarian cortical biopsies obtained from polycystic ovary syndrome patients were frozen using a slow freezing-rapid thawing protocol. MAIN OUTCOME MEASURE(S): Primordial and primary follicle viability was assessed with trypan blue staining. Unstained isolated follicles were studied by transmission electron microscopy. Histologic and immunohistochemical analysis of apoptosis was performed on tissue sections. RESULT(S): The percentage of unstained follicles considered live was lower (P=.015) after freezing/thawing (71.9%) than before cryopreservation (87.3%). Transmission electron microscopy on follicles considered live confirmed the lack of ultrastructural damage. After freezing/thawing, tissue morphology was preserved, but immunohistochemical analysis shows a significant increase in the apoptosis process. CONCLUSION(S): Ovarian cortical test fragments combined with trypan blue staining on enzymatically isolated follicles is a useful and quick method of assessing the initial quality and viability of follicles in cryopreserved ovarian tissue. This type of test should be used routinely as quality control in ovarian cryopreservation procedures.

High incidence of chromosomal abnormalities in oocyte donors.

Oocyte donors are chosen among phenotypically normal and fertile women who are not expected to carry any chromosomal abnormality. A high incidence of balanced structural chromosomal rearrangements has been found within oocyte donors. This result raises the question of a possible bias in their recruitment with respect to their familial background and/or personal reproductive history.

How can the genetic risks of embryo donation be minimized? Proposed guidelines of the French Federation of CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme).

Embryo donation is now an acceptable practice which offers new possibilities to many infertile couples wishing to procreate. In France, embryo donation, like gamete donation, is controlled by law, but its application has been poorly developed because too many questions remained unsolved and because of the lack of practical guidelines. Here we report the results of the debate which took place within the Genetics Commission of the French Federation of CECOS and the proposed recommendations which followed, emphasizing the genetic background to be considered for embryo donation.