Implication of chromosomal microarray analysis prior to in-utero repair of fetal open neural tube defect.

OBJECTIVE: In-utero repair of open neural tube defects (ONTD) is an accepted treatment option with demonstrated superior outcome for eligible patients. While current guidelines recommend genetic testing by chromosomal microarray analysis (CMA) when a major congenital anomaly is detected prenatally, the requirement for an in-utero repair, based on the Management of Myelomeningocele Study (MOMS) criteria, is a normal karyotype. In this study, we aimed to evaluate if CMA should be recommended as a prerequisite for in-utero ONTD repair. METHODS: This was a retrospective cohort study of pregnancies complicated by ONTD that underwent laparotomy-assisted fetoscopic repair or open-hysterotomy fetal surgery at a single tertiary center between September 2011 and July 2021. All patients met the MOMS eligibility criteria and had a normal karyotype. In a subset of the pregnancies (n = 77), CMA testing was also conducted. We reviewed the CMA results and divided the cohort into two groups according to whether clinically reportable copy-number variants (CNV) were detected (reportable-CNV group) or not (normal-CMA group). Surgical characteristics, complications, and maternal and early neonatal outcomes were compared between the two groups. The primary outcomes were fetal or neonatal death, hydrocephalus, motor function at 12 months of age and walking status at 30 months of age. Standard parametric and non-parametric statistical tests were employed as appropriate. RESULTS: During the study period, 146 fetuses with ONTD were eligible for and underwent in-utero repair. CMA results were available for 77 (52.7%) patients. Of those, 65 (84%) had a normal CMA and 12 (16%) had a reportable CNV, two of which were classified as pathogenic. The first case with a pathogenic CNV was diagnosed with a 749-kb central 22q11.21 deletion spanning low-copy-repeat regions B-D of chromosome 22; the second case was diagnosed with a 1.3-Mb interstitial deletion at 1q21.1q21.2. Maternal demographics, clinical characteristics, operative data and postoperative complications were similar between those with normal CMA results and those with reportable CNVs. There were no significant differences in gestational age at delivery or any obstetric and early neonatal outcome between the study groups. Motor function at birth and at 12 months of age, and walking status at 30 months of age, were similar between the two groups. CONCLUSIONS: Standard diagnostic testing with CMA should be offered when an ONTD is detected prenatally, as this approach has implications for counseling regarding prognosis and recurrence risk. Our results indicate that the presence of a clinically reportable CNV should not a priori affect eligibility for in-utero repair, as overall pregnancy outcome is similar in these cases to that of cases with normal CMA. Nevertheless, significant CMA results will require a case-by-case multidisciplinary discussion to evaluate eligibility. To generalize the conclusion of this single-center series, a larger, multicenter long-term study should be considered. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

No evidence for mutations in NLRP7, NLRP2 or KHDC3L in women with unexplained recurrent pregnancy loss or infertility.

STUDY QUESTION: Are mutations in NLRP2/7 (NACHT, LRR and PYD domains-containing protein 2/7) or KHDC3L (KH Domain Containing 3 Like) associated with recurrent pregnancy loss (RPL) or infertility? SUMMARY ANSWER: We found no evidence for mutations in NLRP2/7 or KHDC3L in unexplained RPL or infertility. WHAT IS KNOWN ALREADY: Mutations in NLRP7 and KHDC3L are known to cause biparental hydatidiform moles (BiHMs), a rare form of pregnancy loss. NLRP2, while not associated with the BiHM pathology, is known to cause recurrent Beckwith Weidemann Syndrome (BWS). STUDY DESIGN, SIZE, AND DURATION: Ninety-four patients with well characterized, unexplained infertility were recruited over a 9-year period from three IVF clinics in Sweden. Blood samples from 24 patients with 3 or more consecutive miscarriages of unknown etiology were provided by the Recurrent Miscarriage Clinic at St Mary's Hospital, London, UK. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were recruited into both cohorts following extensive clinical studies. Genomic DNA was isolated from peripheral blood and subject to Sanger sequencing of NLRP2, NLRP7 and KHDC3L. Sequence electropherograms were analyzed by Sequencher v5.0 software and variants compared with those observed in the 1000 Genomes, single nucleotide polymorphism database (dbSNP) and HapMap databases. Functional effects of non-synonymous variants were predicted using Polyphen-2 and sorting intolerant from tolerant (SIFT). MAIN RESULTS AND THE ROLE OF CHANCE: No disease-causing mutations were identified in NLRP2, NLRP7 and KHDC3L in our cohorts of unexplained infertility and RPL. LIMITATIONS, REASONS FOR CAUTION: Due to the limited patient size, it is difficult to conclude if the low frequency single nucleotide polymorphisms observed in the present study are causative of the phenotype. The design of the present study therefore is only capable of detecting highly penetrant mutations. WIDER IMPLICATIONS OF THE FINDINGS: The present study supports the hypothesis that mutations in NLRP7 and KHDC3L are specific for the BiHM phenotype and do not play a role in other adverse reproductive outcomes. Furthermore, to date, mutations in NLRP2 have only been associated with the imprinting disorder BWS in offspring and there is no evidence for a role in molar pregnancies, RPL or unexplained infertility. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the following sources: Estonian Ministry of Education and Research (Grant SF0180044s09), Enterprise Estonia (Grant EU30020); Mentored Resident research project (Department of Obstetrics and Gynecology, Baylor College of Medicine); Imperial NIHR Biomedical Research Centre; Grant Number C06RR029965 from the National Center for Research Resources (NCCR; NIH). No competing interests declared.

A recurrent intragenic genomic duplication, other novel mutations in NLRP7 and imprinting defects in recurrent biparental hydatidiform moles.

A complete hydatidiform mole (CHM) is an abnormal pregnancy with hyperproliferative vesicular trophoblast and no fetal development. Most CHM are sporadic and androgenetic, but recurrent HM have biparental inheritance (BiHM) with disrupted DNA methylation at differentially methylated regions (DMRs) of imprinted loci. Some women with recurrent BiHM have mutations in the NLRP7 gene on chromosome 19q13.42. Using bisulfite genomic sequencing at eight imprinted DMRs on DNA from two BiHMs, we found a pattern of failure to acquire or maintain DNA methylation at DMRs (PEG3, SNRPN, KCNQ1OT1, GNAS exon 1A) that normally acquire CpG methylation during oogenesis, but not at H19, which acquires CpG methylation during spermatogenesis. Secondary imprints at the GNAS locus showed variable abnormal patterns with both gain and loss of CpG methylation. We found novel missense and splice-site mutations in NLRP7 in women with non-familial recurrent BiHM. We identified and characterized a homozygous intragenic tandem duplication including exons 2 through 5 of NLRP7 that results in a predicted truncated protein in affected women of three unrelated Egyptian kindreds, suggesting a founder effect. Our findings firmly establish that NLRP7 mutations are a major cause of BiHM and confirm presence of a complex pattern of imprinting abnormalities in BiHM tissues.

Biparental hydatidiform moles: a maternal effect mutation affecting imprinting in the offspring.

Highly recurrent hydatidiform moles (HMs) studied to date are not androgenetic but have biparental genomic contribution (BiHM). Affected women have an autosomal recessive mutation that causes their pregnancies to develop into HM. Although there is genetic heterogeneity, a major locus maps to chromosome 19q13.42, but a mutated gene has not yet been identified. Molecular studies have shown that maternal imprinting marks are deregulated in the BiHM trophoblast. The mutations that cause this condition are, therefore, hypothesized to occur in genes that encode transacting factors required for the establishment of imprinting marks in the maternal germline or for their maintenance in the embryo. Although only DNA methylation marks at imprinted loci have been studied in the BiHM, the mutation may affect genes that are essential for other forms of chromatin remodelling at imprinted loci and necessary for correct maternal allele-specific DNA methylation and imprinted gene expression. Normal pregnancies interspersed with BiHM have been reported in some of the pedigrees, but affected women repeatedly attempting pregnancy should be counselled about the risk for invasive trophoblastic disease with each subsequent BiHM.

Tooth enamel defects in mice with a deletion at the Arhgap 6/Amel X locus.

The amelogenin proteins regulate enamel mineral formation in the developing tooth. The human AMELX gene, which encodes the amelogenin proteins, is located within an intron of the Arhgap 6 gene. ARHGAP 6 encodes a Rho GAP, which regulates activity of Rho A, a small G protein involved in intracellular signal transduction. Mice were generated in which the entire ARHGAP 6 gene was deleted by Cre-mediated recombination, which also removed the nested Amel X gene. Enamel from these mice appeared chalky white, and the molars showed excessive wear. The enamel layer was hypoplastic and non-prismatic, whereas other dental tissues had normal morphology. This phenotype is similar to that reported for Amel X null mice, which have a short deletion that removed the region surrounding the translation initiation site, and resembles some forms of X-linked amelogenesis imperfecta in humans. Analysis of the enamel from the Arhgap 6/Amel X-deleted mice verifies that the Amel X gene is nested within the murine Arhgap 6 gene and shows that removal of the entire Amel X gene leads to a phenotype similar to the earlier Amel X null mouse results, in which no amelogenin protein was detected. However, an unusual layer of aprismatic enamel covers the enamel surface, which may be related to the 1.1-Mb deletion, which included Arhgap 6 in these mice.

Recurrent pregnancy loss due to familial and non-familial habitual molar pregnancy.

OBJECTIVES: To present a series of women with recurrent molar pregnancies, including rare familial cases, and discuss etiology and treatment options. METHODS: We performed a detailed clinical evaluation and pedigree analysis of five Egyptian women with recurrent pregnancy loss due to molar pregnancy. RESULTS: The women had a history of four to nine consecutive hydatidiform moles but of no viable pregnancies. Two of the women had molar pregnancies with different husbands who themselves had viable offspring from previous wives; and three of them, who belonged to a family with extensive intermarriage, had a pedigree consistent with an autosomal recessive maternal-effect mutation. CONCLUSIONS: Recurrent pregnancy loss due to habitual molar pregnancy is uncommon and familial cases are extremely rare. The etiology of this disorder is not well understood but likely results from a maternal-effect mutation. Management options are limited, especially for couples who desire to have their own genetic offspring.

The product of the imprinted gene IPL marks human villous cytotrophoblast and is lost in complete hydatidiform mole.

The IPL/TSSC3 gene is expressed nearly exclusively from the maternal allele, and its protein product acts to limit placental growth in mice. This protein specifically marks Type II trophoblast in the labyrinthine layer of the mouse placenta. To investigate mouse-human homologies, we carried out immunohistochemistry with antibodies against human IPL. There was strong expression of IPL in villous cytotrophoblast of the human placenta, contrasting with complete lack of expression in syncytiotrophoblast. Staining for IPL was weak in cells of the villous mesenchyme and extravillous trophoblast, including the cytotrophoblast columns in the basal plate and the intervillous trophoblast islands. The IPL and p57(KIP2)/CDKN1C genes are closely linked and coordinately imprinted, and immunostaining showed that their protein products are co-expressed in villous cytotrophoblast. However, other cell types, including extravillous cytotrophoblast and cells in various non-placental tissues, expressed p57(KIP2), but not IPL. IPL protein was absent in both of two cases of androgenetic complete hydatidiform mole examined by immunostaining, and IPL mRNA was absent in an additional three cases of this neoplasm examined by northern blotting. In the mouse, Ipl-expressing cells disappear at mid- to late-gestation when placental growth ceases, but persistent IPL mRNA and protein expression was observed throughout human gestation, correlating with the continuous growth of the human placenta. These findings highlight dosage regulation of human IPL by imprinting and, more generally, suggest homology between Type II labyrinthine trophoblast in the mouse and villous cytotrophoblast in humans, both of which are proliferative stem cell-like compartments.

Microphthalmia with linear skin defects (MLS), Aicardi, and Goltz syndromes: are they related X-linked dominant male-lethal disorders?

Gene identification for X-linked dominant sporadic disorders is challenging because no extended families exist that can be studied by linkage analysis. Therefore, classic positional cloning approaches are not possible, and other methods have to be used to search for candidate genes. These conditions present the next challenge for disease-gene identification of Mendelian disorders. The various issues and difficulties involved, such as male lethality, X chromosome inactivation, and analysis of phenotypic similarities among different conditions are illustrated through discussion of three X-linked developmental disorders: microphthalmia with linear skin defects (MLS) syndrome, Aicardi syndrome, and Goltz syndrome (focal dermal hypoplasia).

Mutations in the gene encoding methyl-CpG-binding protein 2 cause Rett syndrome.

Rett syndrome is an X-linked dominant neurodevelopmental disorder primarily affecting girls. About 80% of classic Rett syndrome is caused by mutations in the gene for methyl-CpG-binding protein (MeCP2) in Xq28. MeCP2 links DNA methylation to transcriptional repression, and MECP2 mutations likely cause partial or complete loss of function of the protein, leading to inappropriate transcription of downstream genes at critical times in brain development. More severe and milder variant forms can all be caused by similar mutations. Most classic Rett syndrome patients have random X-chromosome inactivation (XCI), but skewed patterns are present in a few. All asymptomatic or mildly mentally delayed female carriers studied to date have non-random XCI patterns, suggesting that this attenuates the deleterious effects of the MECP2 mutations in these women. The finding of non-random XCI patterns in some patients with very early truncations is consistent with this observation and supports that many mutations could cause partial and not complete loss of function. Our observation that the mutant mRNA is stable in three patients with truncating mutations supports this possibility. Further studies will have to be performed to better understand the functional consequences of MECP2 mutations in RTT.

The human homologue (PEG3) of the mouse paternally expressed gene 3 (Peg3) is maternally imprinted but not mutated in women with familial recurrent hydatidiform molar pregnancies.

OBJECTIVES: We mapped a locus for autosomal recessive molar pregnancies with biparental genomic contribution to chromosome 19q13.4 between D19S924 and D19S890. This 5-Mb region is homologous to proximal mouse chromosome 7 and contains a cluster of Krüppel-type zinc finger genes, including the human homologue of the mouse imprinted genes: the paternally expressed gene 3 (PEG3) and the maternally expressed Zim1 genes. We analyzed the PEG3 gene for mutations in women with familial recurrent hydatidiform moles and to determine its imprinting status in humans. METHODS: We used database searches and screened cDNA libraries to find the complete genomic structure of PEG3. Polymerase chain reaction (PCR) amplification and direct sequencing of coding exons and flanking introns were performed on genomic DNA from the affected women. Allele-specific methylation and expression were studied by methylation-sensitive Southern analysis of a 5' located CpG island and by reverse-transcription PCR of total lymphoblast-derived RNA of normal individuals who were informative for two expressed polymorphisms. RESULTS: We did not detect any mutations in the coding region of PEG3 in the affected women. We observed allele-specific methylation of the CpG island and expression from the paternal allele in two independent informative pedigrees. CONCLUSION: Consistent with the findings in the mouse, the human PEG3 gene is expressed from the paternal allele. Our data support that PEG3 is not mutated in women with familial recurrent hydatidiform moles, although mutations in the regulatory regions that might affect imprinting or transcriptional level of the gene could not be evaluated.

Skewed X inactivation in X-linked disorders.

X chromosome inactivation is a process by which the dosage of proteins transcribed from genes on the X chromosome is equalized between males (XY) and females (XX) through the silencing of most genes on one of the two X chromosomes in females. Although the choice of which of the two X's is inactivated is entirely random, not all women have a 50:50 ratio of cells with one or the other X chromosomes active. A number of different mechanisms lead to extremely skewed ratios and this can result in expression of the phenotype of X-linked recessive disorders in females. Nonrandom X inactivation patterns are also associated with selective female survival in male-lethal X-linked dominant disorders or with variable severity of the phenotype in women carrying X-linked dominant mutations. These features are important for the study and gene identification of X-linked disorders and for counseling of affected families.

Terminal osseous dysplasia and pigmentary defects: clinical characterization of a novel male lethal X-linked syndrome.

We describe a new syndrome of distal limb anomalies and pigmentary skin defects in 10 females of a large, four-generation pedigree. The family was ascertained through a 4-month-old infant girl with multiple anomalies, including hypertelorism, iris colobomas, low-set ears, midface hypoplasia, punched-out pigmentary abnormalities over the face and scalp, generalized brachydactyly, and digital fibromatosis. No affected males were identified in this pedigree. Affected females had a lower than normal male-to-female ratio of liveborn offspring, and some of them also had a history of several miscarriages. These findings, together with a significant variability in the phenotype of the affected females, suggest that this condition is inherited in an X-linked dominant fashion, with prenatal male lethality, and that X-inactivation plays an important role in the phenotypic expression of the disease. The syndrome has been described twice in the literature, but only in sporadic cases; it was therefore not recognized as a mendelian entity. Because the most consistent findings are anomalies of the distal skeleton of the limbs and localized pigmentary abnormalities of the skin, we named the syndrome "terminal osseous dysplasia with pigmentary defects." This condition, though rare, can be added to the small group of male lethal X-linked dominant disorders in humans.

Methyl-CpG-binding protein 2 mutations in Rett syndrome.

The X-linked methyl-CpG-binding protein 2 gene (MECP2) encodes a protein that links DNA methylation to transcriptional repression mediated by histone deacetylases. Mutations in MECP2 have been found in 76% of classic Rett syndrome patients. Favourable nonrandom X chromosome inactivation ameliorates the phenotype.

Influence of mutation type and X chromosome inactivation on Rett syndrome phenotypes.

We screened 71 sporadic and 7 familial Rett syndrome (RTT) patients for MECP2 mutations by direct sequencing and determined the pattern of X chromosome inactivation (XCI) in 39 RTT patients. We identified 23 different disease-causing MECP2 mutations in 54 of 71 (76%) sporadic patients and in 2 of 7 (29%) familial cases. We compared electrophysiological findings, cerebrospinal fluid neurochemistry, and 13 clinical characteristics between patients carrying missense mutations and those carrying truncating mutations. Thirty-one of 34 patients (91%) with classic RTT had random XCI. Nonrandom XCI was associated with milder phenotypes, including a mitigated classic RTT caused by a rare early truncating mutation. Patients with truncating mutations have a higher incidence of awake respiratory dysfunction and lower levels of cerebrospinal fluid homovanillic acid. Scoliosis is more common in patients with missense mutations. These data indicate that different MECP2 mutations have similar phenotypic consequences, and random XCI plays an important role in producing the full phenotypic spectrum of classic RTT. The association of early truncating mutations with nonrandom XCI, along with the fact that chimeric mice lacking methyl-CpG-binding protein 2 (MeCP2) function die during embryogenesis, supports the notion that RTT is caused by partial loss of MeCP2 function.

Terminal osseous dysplasia with pigmentary defects maps to human chromosome Xq27.3-xqter.

We have identified a four-generation family with 10 affected females manifesting one or more of the following features: osseous dysplasia involving the metacarpals, metatarsals, and phalanges leading to brachydactyly, camptodactyly, and other digital deformities; pigmentary defects on the face and scalp; and multiple frenula. There were no affected males. We performed X-inactivation studies on seven affected females, using a methylation assay at the androgen receptor locus; all seven demonstrated preferential inactivation of their maternal chromosomes carrying the mutation, and two unaffected females showed a random pattern. These findings indicate that this disorder is linked to the X chromosome. To map the gene for this disorder, we analyzed DNA from nine affected females and five unaffected individuals, using 40 polymorphic markers evenly distributed throughout the X chromosome. Two-point and multipoint linkage analyses using informative markers excluded most of the X chromosome and demonstrated linkage to a region on the long arm between DXS548 and Xqter. A maximum LOD score of 3.16 at recombination fraction 0 was obtained for five markers mapping to Xq27.3-Xq28. The mapping data should facilitate the identification of the molecular basis of this disorder.

Discordance between fetal RhD typing using molecular methods and neonatal typing with serology.

Polymerase chain reaction (PCR)-based genotyping on amniotic fluid in an RhD-negative alloimmunized woman predicted an RhD-negative fetal blood type. The neonate was RhD-positive and developed hemolytic disease. Discrepant results were also observed on paternal testing. PCR analysis with a different set of primers correctly predicted the RhD-positive fetal and paternal blood type. Use of more than one set of primers and parental testing can avoid some of the problems associated with use of PCR genotyping.

Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.

Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females, with an incidence of 1 in 10,000-15,000 (ref. 2). Patients with classic RTT appear to develop normally until 6-18 months of age, then gradually lose speech and purposeful hand use, and develop microcephaly, seizures, autism, ataxia, intermittent hyperventilation and stereotypic hand movements. After initial regression, the condition stabilizes and patients usually survive into adulthood. As RTT occurs almost exclusively in females, it has been proposed that RTT is caused by an X-linked dominant mutation with lethality in hemizygous males. Previous exclusion mapping studies using RTT families mapped the locus to Xq28 (refs 6,9,10,11). Using a systematic gene screening approach, we have identified mutations in the gene (MECP2 ) encoding X-linked methyl-CpG-binding protein 2 (MeCP2) as the cause of some cases of RTT. MeCP2 selectively binds CpG dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor SIN3A (refs 12,13). In 5 of 21 sporadic patients, we found 3 de novo missense mutations in the region encoding the highly conserved methyl-binding domain (MBD) as well as a de novo frameshift and a de novo nonsense mutation, both of which disrupt the transcription repression domain (TRD). In two affected half-sisters of a RTT family, we found segregation of an additional missense mutation not detected in their obligate carrier mother. This suggests that the mother is a germline mosaic for this mutation. Our study reports the first disease-causing mutations in RTT and points to abnormal epigenetic regulation as the mechanism underlying the pathogenesis of RTT.

Characterization of a novel chromo domain gene in xp22.3 with homology to Drosophila msl-3.

The Drosophila male-specific lethal (MSL) genes regulate transcription from the male X chromosome in a dosage compensation pathway that equalizes X-linked gene expression in males and females. The members of this gene family, including msl-1, msl-2, msl-3, mle, and mof, encode proteins with no sequence homology. However, mutations in each of these genes produce a similar phenotype: sex-specific lethality of male embryos caused by the failure of mutants to increase transcription from the single male X chromosome. The MSL gene products assemble into a multiprotein transcriptional activation complex at hundreds of sites along the chromatin of the X chromosome. Here we report the isolation and characterization of a human gene, named MSL3L1, that encodes a protein with significant homology to Drosophila MSL-3 in three distinct regions, including two putative chromo domains. MSL3L1 was identified by database queries with genomic sequence from BAC GS-590J6 (GenBank AC0004554) in Xp22.3 and was evaluated as a candidate gene for several developmental disorders mapping to this region, including OFD1 and SED tarda, as well as Aicardi syndrome and Goltz syndrome.