Jean-Pierre Siffroi

In Vitro Fertilization May Increase the Risk of Beckwith-Wiedemann Syndrome Related to the Abnormal Imprinting of the KCNQ1OT Gene

To the Editor: “Parental imprinting” refers to an epigenetic marking of genes that results in monoallelic expression. This phenomenon plays a critical role in embryogenesis and development. The epigenetic modification of the genome involves methylation changes and the remodeling of chromatin-associated proteins (Li 2002). Imprints are established during the development of germ cells, and the reprogramming of imprinting occurs within the first days after fertilization (Reik and Walter 2001). The alteration of normal imprinting patterns is implicated in a number of human genetic diseases. Among them, the Beckwith-Wiedemann syndrome (BWS [MIM 130650]) is an overgrowth syndrome secondary to the dysregulation of the imprinted 11p15 region (Maher and Reik 2000). Numerous mechanisms are involved in BWS, and ∼70% of cases of BWS are related to epigenetic abnormalities at the 11p15 locus, mostly demethylation of the KvDMR1 region of the KCNQ1OT (previously called “LIT1”) gene (MIM 604115) (Engel et al. 2000; Bliek et al. 2001; Gaston et al. 2001; Weksberg et al. 2001; DeBaun et al. 2002). KCNQ1OT encodes a noncoding antisense transcript within intron 10 of the KCNQ1 gene (MIM 192500) (Lee et al. 1999; Mitsuya et al. 1999; Smilinich et al. 1999) and might be involved in the regulation of parental imprinting of the centromeric domain of the 11p15 region (Fitzpatrick et al. 2002). In sheep and cattle, epigenetic abnormalities have been shown to be involved in large offspring syndrome (LOS) (Young et al. 1998). Affected animals exhibit various phenotypes, including large size at birth. In both species, the syndrome is caused by the in vitro exposure of embryos, between fertilization and the blastocyst stage, to various unusual environments. LOS is related to the loss of imprinting of the IGF2 receptor gene (MIM 147280), which ensures internalization and degradation of IGF2 and displays an antiproliferative function (Young et al. 2001). In vitro preimplantation procedures in mice are also responsible for overgrowth, owing to the abnormal expression of various imprinted genes, particularly the genes located at distal chromosome 7 (h19 [MIM 103280] and igf2 [MIM 147470] genes), orthologous to the human 11p15 region (Humpherys et al. 2001; Rideout et al. 2001). In humans, a case of BWS was recently described after in vitro fertilization (IVF) (Olivennes et al. 2001). Moreover, two recent papers (DeBaun et al. 2003; Maher et al. 2003) described an increase in prevalence of assisted reproductive technologies (ART) in patients with BWS. De Baun et al. (2003) reported a sixfold increase (4.6% vs. 0.76% in the general population) and showed that four of the six patients for whom DNA was available exhibited an isolated demethylation of KvDMR1 in the KCNQ1OT gene. Maher et al. (2003) reported a threefold increase (4% vs. 0.997% in the general population) and demonstrated that two of the six patients on whom molecular analysis could be done also exhibited an isolated demethylation of KvDMR1. Our department is a reference center in France for molecular diagnosis of BWS, and patients are referred from various medical departments (neonatology, pediatrics, genetics, and fetopathology). We studied a series of 149 patients referred for overgrowth syndromes and diagnosed as BWS, since all of them exhibited genetic or epigenetic defects at the 11p15 locus. According to the inclusion criteria described elsewhere (Gaston et al. 2001), 102 patients exhibited a complete form of BWS, and 47 exhibited an incomplete form of BWS. The techniques used to analyze the 11p15 region have been described elsewhere (Gaston et al. 2000, 2001). Epigenetic changes concerned 104 (70%) patients, most of whom (n=90) exhibited a loss of KvDMR1 methylation. Fourteen patients (9.4%) exhibited isolated hypermethylation of the H19 gene. Forty-two patients exhibited a genetic defect: 11p15 uniparental disomy (n=35; 23.5%) and germline CDKN1C (MIM 600856) mutation (n=7; 4.7%). Three patients (2%) had a chromosomal abnormality. Six of the 149 patients were born following ART. Of note, these six patients exhibited the same epigenetic abnormality (isolated demethylation of KvDMR1 with a demethylation index varying 72%–100%) (fig. 1). All of them were sporadic cases, and one was a DZ twin. The clinical features of these patients and the procedures of ART used for their conception are summarized in table 1. As shown in table 1, the phenotypes of patients born after ART were not different from phenotypes of the other patients (n=84) with isolated demethylation of KVDMR1, with the exception that only one patient born after ART exhibited ear abnormalities. These children were issued from various ART procedures: classical IVF, intracytoplasmic sperm injection (ICSI), embryo freezing, and transfer on day 2, day 3, and day 5. More recent procedures, like ICSI (two of six patients) or blastocyst transfer (one of six patients), did not prevail over other techniques. The representation of ART (4%) in our series is three times higher than that in the general population (1.3%), according to the national report of the French Ministry of Health (9,930 of 770,000 live births resulting in 1,999 from IVF, ICSI, or frozen embryo transfer). On the basis of this report, we would have expected 1.94 of the 149 patients with BWS to be born as a result of ART. To test the significance of this difference of frequencies, we used the Fisher’s exact test (P=.01) as well as the Poisson approximation (two-tailed P=.018; 95% CI 1.5–8.7). Strength of the association between exposure to ART and risk of BWS is expressed by an odds ratio of 3.2 (95% CI 1.4–7.3). This rate is the same as that described by Maher et al. (2003) but lower than that described by DeBaun et al. (2003), which addressed a prospective study. In our series and in Maher’s series, this rate is probably underestimated, as specific questions regarding ART have only been asked systematically in the past year. Figure 1 Methylation analysis of KvDMR1 in liver tissue (patient 131) and leukocytes (patients 15, 94, 98, 115, and 137) from the six patients with BWS born after ART and in leukocytes from a normal control (C). Genomic DNA was digested with BamHI and the methylation-sensitive ... Table 1 Clinical Characteristics of the Six Patients with BWS Born Following ART Although the analysis of the imprinting status at chromosome 15q11-13 in children born after ICSI did not reveal an imprinting defect (Manning et al. 2000), two recent papers reported three patients with Angelman syndrome (MIM 105830) born after ICSI (Cox et al. 2002; Orstavik et al. 2002). All three patients exhibited an imprinting defect, which is a rare cause of Angelman syndrome. As in the previous two reports (DeBaun et al. 2003; Maher et al. 2003), our data suggest that ART may favor imprinting alterations at the centromeric imprinted 11p15 locus and, consequently, the incidence of BWS. These data highlight the need to carefully follow up children born after ART to test for BWS and other diseases related to imprinted regions. Although no specific procedures of ART appear to be associated with a risk of BWS in our series, these data lend support to the importance of precisely recording these different procedures of ART, particularly the stimulation protocol, the biological technique, the stage of maturation of the gametes, the culture media used at each step, and the timing of embryo transfer.

KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes

KIF7, the human ortholog of Drosophila Costal2, is a key component of the Hedgehog signaling pathway. Here we report mutations in KIF7 in individuals with hydrolethalus and acrocallosal syndromes, two multiple malformation disorders with overlapping features that include polydactyly, brain abnormalities and cleft palate. Consistent with a role of KIF7 in Hedgehog signaling, we show deregulation of most GLI transcription factor targets and impaired GLI3 processing in tissues from individuals with KIF7 mutations. KIF7 is also a likely contributor of alleles across the ciliopathy spectrum, as sequencing of a diverse cohort identified several missense mutations detrimental to protein function. In addition, in vivo genetic interaction studies indicated that knockdown of KIF7 could exacerbate the phenotype induced by knockdown of other ciliopathy transcripts. Our data show the role of KIF7 in human primary cilia, especially in the Hedgehog pathway through the regulation of GLI targets, and expand the clinical spectrum of ciliopathies.

Human Male Infertility Associated with Mutations in NR5A1 Encoding Steroidogenic Factor 1

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%-50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of NR5A1 have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in NR5A1 cause male infertility, we sequenced NR5A1 in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in NR5A1. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. NR5A1 mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

Screening for Genomic Rearrangements and Methylation Abnormalities of the 15q11-q13 Region in Autism Spectrum Disorders

BACKGROUND Maternally derived duplications of the 15q11-q13 region are the most frequently reported chromosomal aberrations in autism spectrum disorders (ASD). Prader-Willi and Angelman syndromes, caused by 15q11-q13 deletions or abnormal methylation of imprinted genes, are also associated with ASD. However, the prevalence of these disorders in ASD is unknown. The aim of this study was to assess the frequency of 15q11-q13 rearrangements in a large sample of patients ascertained for ASD. METHODS A total of 522 patients belonging to 430 families were screened for deletions, duplications, and methylation abnormalities involving 15q11-q13 with multiplex ligation-dependent probe amplification (MLPA). RESULTS We identified four patients with 15q11-q13 abnormalities: a supernumerary chromosome 15, a paternal interstitial duplication, and two subjects with Angelman syndrome, one with a maternal deletion and the other with a paternal uniparental disomy. CONCLUSIONS Our results show that abnormalities of the 15q11-q13 region are a significant cause of ASD, accounting for approximately 1% of cases. Maternal interstitial 15q11-q13 duplications, previously reported to be present in 1% of patients with ASD, were not detected in our sample. Although paternal duplications of chromosome 15 remain phenotypically silent in the majority of patients, they can give rise to developmental delay and ASD in some subjects, suggesting that paternally expressed genes in this region can contribute to ASD, albeit with reduced penetrance compared with maternal duplications. These findings indicate that patients with ASD should be routinely screened for 15q genomic imbalances and methylation abnormalities and that MLPA is a reliable, rapid, and cost-effective method to perform this screening.

Prospective diagnostic analysis of copy number variants using SNP microarrays in individuals with autism spectrum disorders.

Copy number variants (CNVs) have repeatedly been found to cause or predispose to autism spectrum disorders (ASDs). For diagnostic purposes, we screened 194 individuals with ASDs for CNVs using Illumina SNP arrays. In several probands, we also analyzed candidate genes located in inherited deletions to unmask autosomal recessive variants. Three CNVs, a de novo triplication of chromosome 15q11–q12 of paternal origin, a deletion on chromosome 9p24 and a de novo 3q29 deletion, were identified as the cause of the disorder in one individual each. An autosomal recessive cause was considered possible in two patients: a homozygous 1p31.1 deletion encompassing PTGER3 and a deletion of the entire DOCK10 gene associated with a rare hemizygous missense variant. We also identified multiple private or recurrent CNVs, the majority of which were inherited from asymptomatic parents. Although highly penetrant CNVs or variants inherited in an autosomal recessive manner were detected in rare cases, our results mainly support the hypothesis that most CNVs contribute to ASDs in association with other CNVs or point variants located elsewhere in the genome. Identification of these genetic interactions in individuals with ASDs constitutes a formidable challenge.

Lack of Association between Genetic Polymorphisms in Enzymes Associated with Folate Metabolism and Unexplained Reduced Sperm Counts

Background The metabolic pathway of folate is thought to influence DNA stability either by inducing single/double stranded breaks or by producing low levels of S-adenosyl-methionine leading to abnormal gene expression and chromosome segregation. Polymorphisms in the genes encoding enzymes in the folate metabolism pathway show distinct geographic and/or ethnic variations and in some cases have been linked to disease. Notably, the gene Methylenetetrahydrofolate reductase (MTHFR) in which the homozygous (TT) state of the polymorphism c.665C>T (p.A222V) is associated with reduced specific activity and increased thermolability of the enzyme causing mild hyperhomocysteinemia. Recently several studies have suggested that men carrying this polymorphism may be at increased risk to develop infertility. Methodology/Principal Findings We have tested this hypothesis in a case/control study of ethnic French individuals. We examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V and E429A), Methionine synthase reductase MTRR; (I22M and S175L) and Cystathionine beta-synthase (CBS; G307S). The case population consisted of DNA samples from men with unexplained azoospermia (n = 70) or oligozoospermia (n = 182) and the control population consisted of normospermic and fertile men (n = 114). We found no evidence of an association between the incidence of any of these variants and reduced sperm counts. In addition haplotype analysis did not reveal differences between the case and control populations. Conclusions/Significance We could find no evidence for an association between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

Deletion of 9p associated with gonadal dysfunction in 46,XY but not in 46,XX human fetuses

A locus on distal 9p has been reported to be deleted in patients with mental retardation, craniofacial dysmorphic traits, notably trigonocephaly, and a high frequency of genital and/or gonadal anomalies (Alfis syndrome).1 More recently, some cases of 46,XY male to female sex reversal without dysmorphic traits have been described in association with distal deletions of 9p.2–5 46,XY patients described so far have had gonadal anomalies ranging from streak gonads, to partially masculinised dysgenetic gonads, to normal testes. A family of genes containing a unique zinc finger DNA binding domain, which has been called DM, as it is contained in two proteins involved in invertebrate sex determination, doublesex and mab-3, has recently been recognised. Three genes belonging to this family have been identified on 9p24.3, immediately proximal to the centromeric boundary of the minimal region, which is required in two copies for testis development.5 The first gene identified was termed DMRT1 and the two additional DM gene family members are placed in tandem more distally to the breakpoint of the minimal deleted region.6,7 DMRT1 is transcribed only in the embryonic gonads of both sexes and in fetal and adult testis, and is required for postnatal differentiation of both somatic and germ cells in the mouse testis.8 Therefore, this gene is a good candidate to explain the 46,XY sex reversal associated with 9p deletions. Recently, a 1 year old 46,XX female patient with premature ovarian failure and deletion of 9p was reported.9 This led to the suggestion of a role for a gene located on 9p in both male and female gonadal development. DMRT1 represents a good candidate for a function in both sexes as it is expressed in the genital ridge in both sexes in mouse and humans.7,8 Here, we report …

Midline defects in deletion 18p syndrome: clinical and molecular characterization of three patients.

The phenotype of monosomy 18p varies widely, the main clinical manifestations being mental and growth retardation, and craniofacial dysmorphism. Clinical features also include growth hormone (GH) deficiency, or holoprosencephaly (HPE). Haploinsufficiency for TGIF, mapped to 18p11.3, is not generally sufficient to cause HPE. To perform a genotype–phenotype correlation, and delineate the region involved in GH deficiency, we carried out a molecular characterization of the 18p deletions, in three patients with midline defects. Two unrelated children, a 7-month-old girl and a 2-month-old boy had del(18p) syndrome and GH deficiency. In addition, the boy had HPE. HPE genes, SHH, ZIC2, SIX3, and TGIF, were tested by denaturing high-performance liquid chromatography and quantitative multiplex of PCR short fluorescent fragments analyses. A deletion of TGIF was confirmed, without any associated mutation for the tested HPE genes, suggesting the role of other genetic or environmental factors. The third patient was his moderately retarded mother. A set of chromosome 18p-specific BACs clones was used as fluorescence in-situ hybridization probes to define the breakpoints. Recently, it was found that there seem to be a breakpoint cluster in the centromeric region at 18p11.1, which was not observed in our patients. The girl was found to have a deletion of 10.3 Mb, with a breakpoint in 18p11.22. The boy and his mother had a smaller deletion (8 Mb), with a breakpoint in 18p11.23. These findings suggest that the distal region on 18p is involved in the main clinical features, and GH deficiency, in 18p deletions.

Birth after TESE–ICSI in a man with hypogonadotropic hypogonadism and congenital adrenal hypoplasia linked to a DAX-1 (NR0B1) mutation

DAX1/NR0B1 mutations are responsible for X-linked congenital adrenal hypoplasia (AHC) associated with hypogonadotropic hypogonadism (HH). Few data are available concerning testicular function and fertility in men with DAX1 mutations. Azoospermia as well as failure of gonadotrophin treatment have been reported. We induced spermatogenesis in a patient who has a DAX1 mutation (c.1210C>T), leading to a stop codon in position 404 (p.Gln404X). His endocrine testing revealed a low testosterone level at 1.2 nmol/l (N: 12–40) with low FSH and LH levels at 2.1 IU/l (N: 1–5 IU/l) and 0.1 IU/l (N: 1–4 IU/l), respectively. Baseline semen analysis revealed azoospermia. Menotropin (Menopur®:150 IU, three times weekly) and human chorionic gonadotrophin (1500 IU, twice weekly) were used. After 20 months of treatment, as azoospermia persisted, bilateral multiple site testicular biopsies were performed. Histology revealed severe hypospermatogenesis. Rare spermatozoa were extracted from the right posterior fragment and ICSI was performed. Four embryos were obtained and, after a frozen–thawed single-embryo transfer, the patients wife became pregnant and gave birth to a healthy boy. We report the first case of paternity after TESE–ICSI in a patient with DAX1 mutation, giving potential hope to these patients to father non-affected children. Furthermore, this case illustrates the fact that patients with X-linked AHC have a primary testicular defect in addition to HH.

Polymorphisms in MTHFR and MTRR genes associated with blood plasma homocysteine concentration and sperm counts

OBJECTIVE To investigate the relationship between MTHFR and MTRR genetic variants with respect to both blood plasma homocysteine concentration and sperm counts. DESIGN Polymerase chain reaction followed by specific enzymatic digestion to determine the genotype of the individuals and blood plasma homocysteine quantification by high-performance liquid chromatography. SETTING Research laboratory. PATIENT(S) Two hundred sixty-eight men seeking infertility counseling and 254 partners of infertile women. INTERVENTION(S) We studied three MTHFR (c.1286A → C, c.665C → T and c.203G → A) and two MTRR (c.66A → G and c.524C → T) single-nucleotide polymorphisms and characterized sperm parameters in both oligozoospermic and normospermic men. A cohort of 522 men was examined for this study. A subgroup of 103 men was constituted for quantification of Hcy levels. MAIN OUTCOME MEASURE(S) Semen samples were collected for determinations of sperm concentration, motility, and morphology according to World Health Organization guidelines as well as for DNA isolation. Blood samples of the corresponding individuals were obtained to quantify plasma homocysteine levels. RESULT(S) We did not observe a relationship between homocysteinemia and sperm counts. The MTHFR c.665C → T variant is associated with mild hyperhomocysteinemia in blood plasma in the TT homozygous state. CONCLUSION(S) No association was found between MTHFR/MTRR genetic variants and sperm counts. Although no association was observed with reduced sperm counts, the MTHFR 665TT genotype is associated with a significant increase in blood plasma homocysteine levels.