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Pathogenic variants in LRBA, encoding the LPS Responsive Beige-Like Anchor (LRBA) protein, are responsible for recessive, early-onset hypogammaglobulinemia, severe multi-organ autoimmunity, and lymphoproliferation, with increased risk for malignancy. LRBA deficiency has a wide clinical spectrum with variable age of onset and disease severity. Three apparently unrelated patients with LRBA deficiency, of Georgian Jewish descent, were homozygous for LRBA c.6640C T, p.R2214*, leading to a stop upstream of the LRBA BEACH domain. Despite carrying the same LRBA genotype, the three patients differed in clinical course: the first patient was asymptomatic until age 25 years; the second presented with failure to thrive at age 3 months; and the third presented at age 7 years with immune cytopenias and severe infections. Two of the patients developed malignancies: the first patient was diagnosed with recurrent Hodgkin's disease at age 36 years, and the second patient developed aggressive gastric cancer at age 15 years. Among Georgian Jews, the carrier frequency of the LRBA p.R2214* allele was 1.6% (4 of 236 Georgian Jewish controls). The allele was absent from other populations. Haplotype analysis showed a shared origin of the mutation. These three patients revealed a pathogenic LRBA founder allele in the Georgian Jewish population, support the diverse and complex clinical spectrum of LRBA deficiency, and support the possibility that LRBA deficiency predisposes to malignancy.

ObjectiveTo determine the yield of genetic diagnoses using chromosomal microarray (CMA) and trio whole exome sequencing (WES), separately and combined, among patients with cryptogenic cerebral palsy (CP).MethodsTrio WES of patients with prior CMA analysis for cryptogenic CP, defined as disabling, non-progressive motor symptoms beginning before the age of 3 years without known cause.ResultsGiven both CMA analysis and trio WES, clinically significant genetic findings were identified for 58% of patients (26 of 45). Diagnoses were eight large CNVs detected by CMA and 18 point mutations detected by trio WES. None had more than one severe mutation. Approximately half of events (14 of 26) were de novo. Yield was significantly higher in patients with CP with comorbidities (69%, 22 of 32) than in those with pure motor CP (31%, 4 of 13; p=0.02). Among patients with genetic diagnoses, CNVs were more frequent than point mutations among patients with congenital anomalies (OR 7.8, 95% CI 1.2 to 52.4) or major dysmorphic features (OR 10.5, 95% CI 1.4 to 73.7). Clinically significant mutations were identified in 18 different genes: 14 with known involvement in CP-related disorders and 4 responsible for other neurodevelopmental conditions. Three possible new candidate genes for CP were ARGEF10, RTF1 and TAOK3.ConclusionsCryptogenic CP is genetically highly heterogeneous. Genomic analysis has a high yield and is warranted in all these patients. Trio WES has higher yield than CMA, except in patients with congenital anomalies or major dysmorphic features, but these methods are complementary. Patients with negative results with one approach should also be tested by the other.

Purpose We previously developed Haploseek, a method for comprehensive preimplantation genetic testing (PGT). However, some key features were missing, and the method has not yet been systematically validated. Methods We extended Haploseek to incorporate DNA from embryo grandparents and to allow testing of variants on chromosome X or in regions where parents share common haplotypes. We then validated Haploseek on 151 embryo biopsies from 27 clinical PGT cases. We sequenced all biopsies to low coverage (0.2×), and performed single-nucleotide polymorphism (SNP) microarray genotyping on the embryos' parents and siblings/grandparents. We used the extended Haploseek to predict chromosome copy-number variants (CNVs) and relevant variant-flanking haplotypes in each embryo. We validated haplotype predictions for each clinical sample against polymerase chain reaction (PCR)-based PGT case results, and CNV predictions against established commercial kits. Results For each of the 151 embryo biopsies, all Haploseek-derived haplotypes and CNVs were concordant with clinical PGT results. The cases included 17 autosomal dominant, 5 autosomal recessive, and 3 X-linked monogenic disorders. In addition, we evaluated 1 Robertsonian and 2 reciprocal translocations, and 17 cases of chromosome copy-number counting were performed. Conclusion Our results demonstrate that Haploseek is clinically accurate and fit for all standard clinical PGT applications.

Background Deficiency in Transaldolase, an essential enzyme in regulating NADPH and ribose 5-phosphate production, has been reported in only 39 patients to date. Most patients present already prenatally/neonatally with intra uterine growth retardation, hepato(spleno)megaly and consequent liver failure, anemia, thrombocytopenia, dysmorphic facial features, cardiac and skin abnormalities and hypergonadotropic hypogonadism in cases of late-onset presentation. The mechanism underlying gonadal dysfunction is not fully understood. Clinical case, methods and results A 15y old male from an Indian-Jewish background, with atrial-septal-defect, renal tubulopathy, mild facial dysmorphism and transient liver enzymes abnormalities presented with absence of pubertal development. His gonadotropins levels were elevated (LH – 22 IU/L, FSH 103 IU/L) while testosterone was low. Whole exome sequencing revealed homozygous variant in a novel T167M missense mutation in TALDO1 (transaldolase) gene whereas both parents were heterozygote carriers. Proteine structure analysis indicate that Threonine 167 is part of transaldolase catalytic site and critical for its function. As transaldolase activity is required for nucleic acids production, NADPH synthesis and reduction of cellular Reactive Oxygen Species (ROS) we measured the ROS accumulation using 2′,7′-dichlorodihydrofluorescein diacetate, in fibroblasts derived from the patient and normal controls. The affected patient's fibroblasts showed a significantly increased accumulation of ROS (207%, P=0.007), compared to controls indicating an increased susceptibility to oxidative stress. Conclusion The novel T167M missense mutation in the transaldolase (TALDO1) gene causes a unique clinical presentation including a relative mild liver involvement and hypergonadotrophic hypogonadism. The compromised TALDO1 activity in preventing cellular ROS accumulation in actual patient's fibroblasts is shown here for the first time and may indicate the therapeutic use of antioxidants in all patients with TALDO1 dysfunction. The high expression of TALDO1 in the testis and the gonadal failure suggests its tissue specific requirement in preventing the accumulation of ROS during steroidogenesis. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Deficiency of the endoplasmic reticulum transmembrane protein ARV1 leads to epileptic encephalopathy in humans and in mice. ARV1 is highly conserved, but its function in human cells is unknown. Studies of yeast arv1 null mutants indicate that it is involved in a number of biochemical processes including the synthesis of sphingolipids and glycosylphosphatidylinositol (GPI), a glycolipid anchor that is attached to the C-termini of many membrane bound proteins. GPI anchors are post-translational modifications, enabling proteins to travel from the endoplasmic reticulum (ER) through the Golgi and to attach to plasma membranes. We identified a homozygous pathogenic mutation in ARV1, p.Gly189Arg, in two brothers with infantile encephalopathy, and characterized the biochemical defect caused by this mutation. In addition to reduced expression of ARV1 transcript and protein in patients' fibroblasts, complementation tests in yeast showed that the ARV1 p.Gly189Arg mutation leads to deficient maturation of Gas1, a GPI-anchored protein, but does not affect sphingolipid synthesis. Our results suggest, that similar to mutations in other proteins in the GPI-anchoring pathway, including PIGM, PIGA, and PIGQ, ARV1 p.Gly189Arg causes a GPI anchoring defect and leads to early onset epileptic encephalopathy.

Mutations in the gene HSD17B3 encoding the 17-beta hydroxysteroid dehydrogenase 3 enzyme cause testosterone insufficiency leading to XY disorders of sex development. In this study the clinical and molecular characteristics of three patients from consanguineous families are elucidated. We identified three patients from two unrelated families with XY DSD and a novel homozygous HSD17B3:c. 673G>A mutation. The effect of the mutation on splicing was determined in RNA extracted from the testis of one patient. Three patients presented at ages 0.1, 8 and 0.7 years with ambiguous genitalia and an XY Karyotype. Endocrine workup showed normal cortisol and mineralocorticoid levels with a low testosterone/androstenedione ratio. Whole-exome sequencing, carried out in the first family, revealed a homozygous novel mutation in the HSD17B3 gene: c. 673G>A, p. V225M. The same mutation was found by Sanger sequencing in the third unrelated patient. Haplotype analysis of a 4 Mb region surrounding the HSD17B3 gene on chromosome 9 revealed that the mutation resides on the same allele in all three patients. The mutation, being the first nucleic acid on exon 10, affects splicing and causes exon 10 skipping in one of our patients’ testes. The novel homozygous c. 673G>A, p. V225M mutation in the 17HSDB3 gene is likely a founder mutation and causes severe XY-DSD. It changes a conserved amino acid residue, and also alters 17HSDB3 gene transcription by causing skipping of exon 10, thereby contributing to an imbalance in the relevant protein isoforms and consequently, significant decreased 17HDSB3 enzymatic activity.

Processing of Precursor RNA 1 (POP1) is a core protein component shared by two essential closely related eukaryotic ribonucleoprotein complexes: RNase MRP (the mitochondrial RNA processing ribonuclease) and RNase P. Recently, five patients harboring mutations in POP1 have been reported with severe spondylo-epi-metaphyseal dysplasia and extremely short stature. We report a unique clinical phenotype resulting from the novel homozygous R211Q POP1 mutation in three patients from one family, presenting with severe short stature but only subtle skeletal dysplastic changes that are merely metaphyseal. The RNA moiety of the RNase-MRP complex quantified in RNA extracted from peripheral lymphocytes was dramatically reduced in affected patients indicating instability of the enzymatic complex. However, pre5.8s rRNA, a substrate of RNase-MRP complex, was not accumulated in patients' RNA unlike in the previously reported POP1 mutations; this may explain the uniquely mild phenotype in our cases, and questions the assumption that alteration in ribosomal biogenesis is the pathophysiological basis for skeletal disorders caused by POP1 mutations. Finally, POP1 mutations should be considered in familial cases with severe short stature even when skeletal dysplasia is not strongly evident.

Glycosylphosphatidylinositol (GPI) is synthesized in the endoplasmic reticulum (ER) and added onto proteins to form GPI-anchored proteins. Among the many proteins involved in this process, ACAT-related enzyme-2 required for viability 1 (Arv1) is a candidate, functioning as a flippase that translocates GPI intermediates from the cytoplasmic side into the luminal side of the ER membranes. Here, we show that the deletion of the ARV1 gene in yeast leads to cold-sensitive defects in cell growth and GPI anchor synthesis. Furthermore, complementation assays show that the overexpression of a missense human ARV1-G189R mutant does not completely restore the cold-sensitive phenotypes of the yeast arv1 mutant. Our results support the proposed role of Arv1 in GPI anchor synthesis and suggest that ARV1-linked human diseases result from defective GPI anchor synthesis.

Mutations in the gene HSD17B3 encoding the 17-beta hydroxysteroid dehydrogenase 3 enzyme cause testosterone insufficiency leading to XY disorders of sex development. In this study the clinical and molecular characteristics of three patients from consanguineous families are elucidated.We identified three patients from two unrelated families with XY DSD and a novel homozygous HSD17B3:c. 673GA mutation. The effect of the mutation on splicing was determined in RNA extracted from the testis of one patient.Three patients presented at ages 0.1, 8 and 0.7 years with ambiguous genitalia and an XY Karyotype. Endocrine workup showed normal cortisol and mineralocorticoid levels with a low testosterone/androstenedione ratio. Whole-exome sequencing, carried out in the first family, revealed a homozygous novel mutation in the HSD17B3 gene: c. 673GA, p. V225M. The same mutation was found by Sanger sequencing in the third unrelated patient. Haplotype analysis of a 4 Mb region surrounding the HSD17B3 gene on chromosome 9 revealed that the mutation resides on the same allele in all three patients. The mutation, being the first nucleic acid on exon 10, affects splicing and causes exon 10 skipping in one of our patients' testes.The novel homozygous c. 673GA, p. V225M mutation in the 17HSDB3 gene is likely a founder mutation and causes severe XY-DSD. It changes a conserved amino acid residue, and also alters 17HSDB3 gene transcription by causing skipping of exon 10, thereby contributing to an imbalance in the relevant protein isoforms and consequently, significant decreased 17HDSB3 enzymatic activity.

Mutations in Vaccinia-related kinase 1 (VRK1) have emerged as a cause of severe neuronal phenotypes in human, including brain developmental defects and degeneration of spinal motor neurons, leading to Spinal Muscular Atrophy (SMA) or early onset Amyotrophic Lateral Sclerosis (ALS). Vrk1 gene-trap partial Knockout (KO) mice (Vrk1GT3/GT3), which express decreased levels of Vrk1, are sterile due to impaired gamete production. Here, we examined whether this mouse model also presents neuronal phenotypes. We found a 20–50% reduction in Vrk1 expression in neuronal tissues of the Vrk1GT3/GT3 mice, leading to mild neuronal phenotypes including significant but small reduction in brain mass and motor (rotarod) impairment. Analysis of gene expression in the Vrk1GT3/GT3 cortex predicts novel roles for VRK1 in neuronal pathways including neurotrophin signaling, axon guidance and pathways implicated in the pathogenesis of ALS. Together, our studies of the partial KO Vrk1 mice reveal that even moderately reduced levels of Vrk1 expression result in minor neurological impairment and indicate new neuronal pathways likely involving VRK1.

Breast cancer among Palestinian women has lower incidence than in Europe or North America, yet is very frequently familial. We studied genetic causes of this familial clustering in a consecutive hospital-based series of 875 Palestinian patients with invasive breast cancer, including 453 women with diagnosis by age 40, or with breast or ovarian cancer in a mother, sister, grandmother, or aunt (“discovery series”); and 422 women diagnosed after age 40 and with negative family history (“older-onset sporadic patient series”). Genomic DNA from women in the discovery series was sequenced for all known breast cancer genes, revealing a pathogenic mutation in 13% (61/453) of patients. These mutations were screened in all patients and in 300 Palestinian female controls, revealing 1.0% (4/422) carriers among older, non-familial patients and two carriers among controls. The mutational spectrum was highly heterogeneous, including pathogenic mutations in eleven different genes: BRCA1, BRCA2, TP53, ATM, CHEK2, BARD1, BRIP1, PALB2, MRE11A, PTEN, and XRCC2. BRCA1 carriers were significantly more likely than other patients to have triple negative tumors (P = 0.03). The single most frequent mutation was TP53 p.R181C, which was significantly enriched in the discovery series compared to controls (P = 0.01) and was responsible for 15% of breast cancers among young onset or familial patients. TP53 p.R181C predisposed specifically to breast cancer with incomplete penetrance, and not to other Li-Fraumeni cancers. Palestinian women with young onset or familial breast cancer and their families would benefit from genetic analysis and counseling. This article is protected by copyright. All rights reserved.

The causes of ovarian dysgenesis remain incompletely understood. Two sisters with XX ovarian dysgenesis carried compound heterozygous truncating mutations in the BRCA2 gene that led to reduced BRCA2 protein levels and an impaired response to DNA damage, which resulted in chromosomal breakage and the failure of RAD51 to be recruited to double-stranded DNA breaks. The sisters also had microcephaly, and one sister was in long-term remission from leukemia, which had been diagnosed when she was 5 years old. Drosophila mutants that were null for an orthologue of BRCA2 were sterile, and gonadal dysgenesis was present in both sexes. These results revealed a new role for BRCA2 and highlight the importance to ovarian development of genes that are critical for recombination during meiosis. (Funded by the Israel Science Foundation and others.).

Recessive CRB2 mutations were recently reported to cause both steroid resistant nephrotic syndrome and prenatal onset ventriculomegaly with kidney disease. We report two Ashkenazi Jewish siblings clinically diagnosed with ciliopathy. Both presented with severe congenital hydrocephalus and mild urinary tract anomalies. One affected sibling also has lung hypoplasia and heart defects. Exome sequencing and further CRB2 analysis revealed that both siblings are compound heterozygotes for CRB2 mutations p.N800K and p.Gly1036Alafs*43, and heterozygous for a deleterious splice variant in the ciliopathy gene TTCB21. CRB2 is a polarity protein which plays a role in ciliogenesis and ciliary function. Biallelic CRB2 mutations in animal models result in phenotypes consistent with ciliopathy. This report expands the phenotype of CRB2 mutations to include lung hypoplasia and uretero-pelvic renal anomalies, and confirms cardiac malformation as a feature. We suggest that CRB2-associated disease is a new ciliopathy syndrome with possible digenic/triallelic inheritance, as observed in other ciliopathies. Clinically, CRB2 should be assessed when ciliopathy is suspected, especially in Ashkenazi Jews, where we found that p.N800K carrier frequency is 1 of 64. Patients harboring CRB2 mutations should be tested for the complete range of ciliopathy manifestations.

To identify the genetic basis of a recessive syndrome characterized by prenatal hyperechogenic brain foci, congenital microcephaly, hypothalamic midbrain dysplasia, epilepsy, and profound global developmental disability.Identification of the responsible gene by whole exome sequencing and homozygosity mapping.Ten patients from 4 consanguineous Palestinian families manifested in utero with hyperechogenic brain foci, microcephaly, and intrauterine growth retardation. Postnatally, patients had progressive severe microcephaly, neonatal seizures, and virtually no developmental milestones. Brain imaging revealed dysplastic elongated masses in the midbrain-hypothalamus-optic tract area. Whole exome sequencing of one affected child revealed only PCDH12 c.2515CT, p.R839X, to be homozygous in the proband and to cosegregate with the condition in her family. The allele frequency of PCDH12 p.R839X is0.00001 worldwide. Genotyping PCDH12 p.R839X in 3 other families with affected children yielded perfect cosegregation with the phenotype (probability by chance is 2.0 × 10(-12)). Homozygosity mapping revealed that PCDH12 p.R839X lies in the largest homozygous region (11.7 MB) shared by all affected patients. The mutation reduces transcript expression by 84% (p2.4 × 10(-13)). PCDH12 is a vascular endothelial protocadherin that promotes cellular adhesion. Endothelial adhesion disruptions due to mutations in OCLN or JAM3 also cause congenital microcephaly, intracranial calcifications, and profound psychomotor disability.Loss of function of PCDH12 leads to recessive congenital microcephaly with profound developmental disability. The phenotype resembles Aicardi-Goutières syndrome and in utero infections. In cases with similar manifestations but no evidence of infection, our results suggest consideration of an additional, albeit rare, cause of congenital microcephaly.

ObjectiveTo identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy.MethodsIdentification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients’ cells and in yeast.ResultsTwo brothers from a consanguineous Palestinian family presented with progressive spinocerebellar ataxia, mental retardation and psychotic episodes. Serial brain imaging showed severe progressive cerebellar atrophy. Whole exome sequencing revealed a novel mutation: pitrilysin metallopeptidase 1 (PITRM1) c.2795C>T, p.T931M, homozygous in the affected children and resulting in 95% reduction in PITRM1 protein. Whole genome sequencing revealed a chromosome X structural rearrangement that also segregated with the disease. Independently, two siblings from a second Palestinian family presented with similar, somewhat milder symptoms and the same PITRM1 mutation on a shared haplotype. PITRM1T931M carrier frequency was 0.027 (3/110) in the village of the first family evaluated, and 0/300 among Palestinians from other locales. PITRM1 is a mitochondrial matrix enzyme that degrades 10–65 amino acid oligopeptides, including the mitochondrial fraction of amyloid-beta peptide. Analysis of peptide cleavage activity by the PITRM1T931M protein revealed a significant decrease in the degradation capacity specifically of peptides ≥40 amino acids.ConclusionPITRM1T931M results in childhood-onset recessive cerebellar pathology. Severity of PITRM1-related disease may be affected by the degree of impairment in cleavage of mitochondrial long peptides. Disruption and deletion of X linked regulatory segments may also contribute to severity.

Prenatal genetic testing is not generally applicable to the very early stages of pregnancy (prior to week 8 gestation), a time period that is crucial to pregnant couples with high risk for transmission of genetic disease to their fetus. Therefore, we developed a new ultra-sensitive targeted next generation sequencing method for noninvasive haplotype-based paternal allele exclusion testing of the cystic fibrosis-associated gene, CFTR. This new method was compared to a conventional library prep and sequencing analysis method and all test results were validated by amniotic fluid testing at later stages of pregnancy. Out of 7 enrolled couples, who provided at least two blood samples (at least one week apart) for noninvasive CFTR testing, a result was obtained for 6 fetuses. Using the new hypersensitive method, all six couples (100%) received a correct diagnosis for the paternal allele as opposed to 3/6 (50%) when tested with the conventional strategy. Among 4 couples who provided just one early pregnancy blood draw for analysis, diagnosis was possible in one fetus, but only using the ultra-sensitive method. Thus, we describe a novel noninvasive CFTR screening method which demonstrates unprecedented fetal allele typing accuracy in the earliest stages of pregnancy.

New technologies are revealing genetic variants of unknown significance (VUS), raising questions about the indications that call for preimplanation genetic diagnosis (PGD). Two couples requesting PGD for VUS are presented. The first couple requested PGD for Lynch syndrome. Whole exome sequencing identified in a healthy male with a family history of Lynch-associated tumours, a MLH1 missense variant. The variant had not been reported as pathogenic, but was predicted as damaging by algorithms. The second couple had a child diagnosed with pervasive developmental disorder and intellectual disability, carrying a microduplication on chr:Xp.22.3, and a microdeletion on chr:17q21.31. The maternally inherited X linked microduplication was also present in the mother's healthy brother and daughter, whereas the chr17 microdeletion was a de-novo event. As chromosomal microarrays and whole-exome sequencing are becoming standard tests, couples are requesting PGD for these VUS. The risk of possible genetic diseases can be reduced by carrying out PGD for uncertain findings, yet will inevitably lead to the birth of affected children despite the transfer of embryos that are not carriers of the familial variants. Findings of unknown significance demand urgent discussion and guidelines for their use as a risk-reduction measure in the preimplantation setting.

BACKGROUND. Noninvasive prenatal testing can be used to accurately detect chromosomal aneuploidies in circulating fetal DNA; however, the necessity of parental haplotype construction is a primary drawback to noninvasive prenatal diagnosis (NIPD) of monogenic disease. Family-specific haplotype assembly is essential for accurate diagnosis of minuscule amounts of circulating cell-free fetal DNA; however, current haplotyping techniques are too time-consuming and laborious to be carried out within the limited time constraints of prenatal testing, hampering practical application of NIPD in the clinic. Here, we have addressed this pitfall and devised a universal strategy for rapid NIPD of a prevalent mutation in the Ashkenazi Jewish (AJ) population. METHODS. Pregnant AJ couples, carrying mutation(s) in GBA, which encodes acid β-glucosidase, were recruited at the SZMC Gaucher Clinic. Targeted next-generation sequencing of GBA-flanking SNPs was performed on peripheral blood samples from each couple, relevant mutation carrier family members, and unrelated individuals who are homozygotes for an AJ founder mutation. Allele-specific haplotypes were constructed based on linkage, and a consensus Gaucher disease–associated founder mutation–flanking haplotype was fine mapped. Together, these haplotypes were used for NIPD. All test results were validated by conventional prenatal or postnatal diagnostic methods. RESULTS. Ten parental alleles in eight unrelated fetuses were diagnosed successfully based on the noninvasive method developed in this study. The consensus mutation–flanking haplotype aided diagnosis for 6 of 9 founder mutation alleles. CONCLUSIONS. The founder NIPD method developed and described here is rapid, economical, and readily adaptable for prenatal testing of prevalent autosomal recessive disease-causing mutations in an assortment of worldwide populations. FUNDING. SZMC, Protalix Biotherapeutics Inc., and Centogene AG.

Summary CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation., Graphical Abstract, Highlights • We identify a disease-associated, differentially methylated region in DM1 hESCs • CTG expansion size correlates with the degree of methylation specifically in DM1 hESCs • DMPK hypermethylation hampers the activity of a regulatory element for SIX5 • DM1 hESCs provide an opportunity to study diseased cardiomyocytes in vitro, Eiges and colleagues used a large cohort of myotonic dystrophy type 1-affected hESCs to characterize a disease-associated, differentially methylated region that hypermethylates as a function of expansion size and, when unmethylated, promotes the expression of a neighboring gene, SIX5, in undifferentiated cells and in in vitro-differentiated cardiomyocytes.

BackgroundFamilial glucocorticoid deficiency (FGD) reflects specific failure of adrenocortical glucocorticoid production in response to adrenocorticotropic hormone (ACTH). Most cases are caused by mutations encoding ACTH-receptor components (MC2R, MRAP) or the general steroidogenesis protein (StAR). Recently, nicotinamide nucleotide transhydrogenase (NNT) mutations were found to cause FGD through a postulated mechanism resulting from decreased detoxification of reactive oxygen species (ROS) in adrenocortical cells.Methods and resultsIn a consanguineous Palestinian family with combined mineralocorticoid and glucocorticoid deficiency, whole-exome sequencing revealed a novel homozygous NNT_c.598 G>A, p.G200S, mutation. Another affected, unrelated Palestinian child was also homozygous for NNT_p.G200S. Haplotype analysis showed this mutation is ancestral; carrier frequency in ethnically matched controls is 1/200. Assessment of patient fibroblasts for ROS production, ATP content and mitochondrial morphology showed that biallelic NNT mutations result in increased levels of ROS, lower ATP content and morphological mitochondrial defects.ConclusionsThis report of a novel NNT mutation, p.G200S, expands the phenotype of NNT mutations to include mineralocorticoid deficiency. We provide the first patient-based evidence that NNT mutations can cause oxidative stress and both phenotypic and functional mitochondrial defects. These results directly demonstrate the importance of NNT to mitochondrial function in the setting of adrenocortical insufficiency.

Background Primary gonadal failure is characterised by primary amenorrhoea or early menopause in females, and oligospermia or azoospermia in males. Variants of the minichromosome maintenance complex component 8 gene (MCM8) have recently been shown to be significantly associated with women’s menopausal age in genome-wide association studies. Furthermore, MCM8-knockout mice are sterile. The objective of this study was to elucidate the genetic aetiology of gonadal failure in two consanguineous families presenting as primary amenorrhoea in the females and as small testes and azoospermia in a male. Methods and results Using whole exome sequencing, we identified two novel homozygous mutations in the MCM8 gene: a splice (c.1954-1G>A) and a frameshift (c.1469-1470insTA). In each consanguineous family the mutation segregated with the disease and both mutations were absent in 100 ethnically matched controls. The splice mutation led to lack of the wild-type transcript and three different aberrant transcripts predicted to result in either truncated or significantly shorter proteins. Quantitative analysis of the aberrantly spliced transcripts showed a significant decrease in total MCM8 message in affected homozygotes for the mutation, and an intermediate decrease in heterozygous family members. Chromosomal breakage following exposure to mitomcyin C was significantly increased in cells from homozygous individuals for c.1954-1G>A, as well as c.1469-1470insTA. Conclusions MCM8, a component of the pre-replication complex, is crucial for gonadal development and maintenance in humans—both males and females. These findings provide new insights into the genetic disorders of infertility and premature menopause in women.

Summary Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5′-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases., Highlights • FMR1 epigenetic gene silencing commonly occurs in the undifferentiated FXS cells • FXS HESCs are heterogeneous for repeat size and methylation levels • This study underscores the importance of multiple HESC lines in disease modeling, Fragile X syndrome (FXS) results from epigenetic silencing of the X-linked FMR1 gene. Using a large set of fragile X-affected human embryonic stem cell lines, Eiges and colleagues show that FMR1 epigenetic gene inactivation commonly occurs prior to embryonic tissue differentiation. This study underscores the importance of examining multiple cell lines in disease modeling, especially in epigenetically regulated disorders like FXS.

Myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults. There are conflicting reports about its effect on female fertility. This study investigated ovarian reserve and IVF-preimplantation genetic diagnosis (PGD) outcome in women with DM1. A total of 21 women undergoing PGD for DM1 were compared with 21 age- and body mass index-matched women undergoing PGD for other diseases. Ovarian reserve markers, response to stimulation, embryo quality and clinical pregnancy and live birth rates were compared. Day-3 FSH concentration was higher, while anti-Mullerian hormone concentration and antral follicle count were lower in the DM1 group (median, range: 6.9 (1.8-11.3) versus 5.7 (1.5-10.7)IU/l; 0.9 (0.17-5.96) versus 2.68 (0.5-9.1)ng/ml; and 13 (0-63) versus 23 (8-40) follicles, respectively, all P < 0.05). Total FSH dose was higher (5200 versus 2250 IU, P = 0.004), while the numbers of oocytes retrieved (10 versus 16, P < 0.04) and metaphase-II oocytes (9 versus 12, P < 0.03) were lower in the DM1 group. The number of cycles with top-quality embryos and the clinical pregnancy rate were lower in the DM1 group. In conclusion, there is evidence of diminished ovarian reserve and less favourable IVF-PGD outcome in women with DM1. Myotonic Dystrophy (DM) is the most common form of muscular dystrophy in adults. There is evidence of subfertility in males affected with the disease but conflicting reports about the effect of the disease on female fertility. The aim of our study was to investigate ovarian reserve and IVF-PGD results in women with DM. Twenty-one women undergoing preimplantation genetic diagnosis (PGD) treatment for DM were compared to 21 age- and BMI matched women undergoing PGD treatment for other diseases. The two groups were compared for antral follicle count (AFC) and serum anti-Mullerian hormone (AMH) levels (the best known markers of ovarian reserve and fertility potential), ovarian response, embryo quality and pregnancy and live birth rates. AFC and the AMH levels were statistically significant lower in the DM group. Total medication dose needed for ovarian stimulation was higher, the number of oocytes and mature oocytes retrieved, and the number of cycles with top quality embryos were lower in the DM group compared to the controls. In conclusion, there is evidence of diminished ovarian reserve, and less favorable IVF-PGD outcome in women with DM. Therefore, we recommend advising these women about the possibility of early decreasing ovarian function in order to prevent any delay in reproductive planning.

Background: Heme oxygenase (HO) is the initial, rate-limiting enzyme in the conversion of heme to bilirubin. Dinucleotide (GT)n repeat length in the promoter region of the encoding gene modulates transcription: shorter alleles, in contrast with longer allele counterparts, are associated with greater gene expression and should result in increased heme catabolism. Objective: We compared the rates of heme catabolism and plasma total bilirubin (TB) between HO-1 promoter genotypes of varying (GT)n repeat lengths in glucose-6-phosphate dehydrogenase (G6PD)-normal and -deficient neonates. Methods:HO-1 promoter length was determined from genomic DNA from previous studies by size discrimination of fluorescently-labeled PCR products with capillary electrophoresis. Sizing was confirmed by sequencing homozygote samples. Alleles were categorized as: short (≤24 GT repeats), medium (25-33 GT repeats), and long (≥34 GT repeats). Previously determined values for blood carboxyhemoglobin, corrected for inspired carbon monoxide (COHbc), and TB were used to determine the rate of heme catabolism and 3rd day TB values for each HO-1 promoter length genotype, respectively. G6PD Mediterranean was determined by PCR analysis. Results: Neither COHbc nor TB values were significantly different between various HO-1 promoter genotypes for either G6PD-normal or -deficient neonates. Conclusions: In the steady state, HO-1 promoter genotypes, based on the length of (GT)n repeats, do not modulate heme catabolism or 3rd day TB values in either G6PD-normal or -deficient neonates.

We previously established that besides its canonical function as E3-ubiquitin ligase, parkin also behaves as a transcriptional repressor of p53. Here we show that parkin differently modulates presenilin-1 and presenilin-2 expression and functions at transcriptional level. Thus, parkin enhances/reduces the protein expression, promoter activity and mRNA levels of presenilin-1 and presenilin-2, respectively, in cells and in vivo. This parkin-associated function is independent of its ubiquitin-ligase activity and remains unrelated to its capacity to repress p53. Accordingly, physical interaction of endogenous or overexpressed parkin with presenilins promoters is demonstrated by chromatin immunoprecipitation assays (ChIP). Furthermore, we identify a consensus sequence, the deletion of which abolishes parkin-dependent modulation of presenilins-1/2 and p53 promoter activities. Interestingly, electrophoretic mobility shift assays (EMSA) revealed a physical interaction between this consensus sequence and wild-type but not mutated parkin. Finally, we demonstrate that the RING1-IBR-RING2 domain of parkin harbors parkin's potential to modulate presenilins promoters. This transcriptional control impacts on presenilins-associated phenotypes, since parkin increases presenilin-1-associated γ-secretase activity and reduces presenilin-2-linked caspase-3 activation. Overall, our data delineate a promoter responsive element targeted by parkin that drives differential regulation of presenilin-1 and presenilin-2 transcription with functional consequences for γ-secretase activity and cell death.

Objective:To identify the genetic basis of a recessive congenital neurologic syndrome characterized by severe hypotonia, arthrogryposis, and respiratory failure.Methods:Identification of the responsible gene by exome sequencing and assessment of the effect of the mutation on protein stability in transfected rat neuronal-like PC12A123.7 cells.Results:Two brothers from a nonconsanguineous Yemeni Jewish family manifested at birth with severe hypotonia and arthrogryposis. The older brother died of respiratory failure at 5 days of age. The proband, now 4.5 years old, has been mechanically ventilated since birth with virtually no milestones achievement. Whole exome sequencing revealed homozygosity of SLC18A3 c.1078G>C, p.Gly360Arg in the affected brothers but not in other family members. SLC18A3 p.Gly360Arg is not reported in world populations but is present at a carrier frequency of 1:30 in healthy Yemeni Jews. SLC18A3 encodes the vesicular acetylcholine transporter (VAChT), which loads newly synthesized acetylcholine from the neuronal cytoplasm into synaptic vesicles. Mice that are VAChT-null have been shown to die at birth of respiratory failure. In human VAChT, residue 360 is located in a conserved region and substitution of arginine for glycine is predicted to disrupt proper protein folding and membrane embedding. Stable transfection of wild-type and mutant human VAChT into neuronal-like PC12A123.7 cells revealed similar mRNA levels, but undetectable levels of the mutant protein, suggesting post-translational degradation of mutant VAChT.Conclusion:Loss of function of VAChT underlies severe arthrogryposis and respiratory failure. While most congenital myasthenic syndromes are caused by defects in postsynaptic proteins, VAChT deficiency is a presynaptic myasthenic syndrome.

The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.

The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism.

Objective To develop a reliable preimplantation genetic diagnosis protocol for antihuman platelet antigen-1 incompatibility for a family in whom antenatal treatment was not possible because of the mother's hypersensitivity to intravenous immunoglobulin (IVIG). Methods Haplotypes were constructed from genomic DNA of the family members. A polymerase chain reaction protocol that included eight microsatellite polymorphic markers and the ITGB3-specific (T196C, rs5918) polymorphism were multiplexed to be used in a single cell protocol, and single blastomeres were analyzed. Results In one preimplantation genetic diagnosis cycle, out of 28 retrieved oocytes, 24 embryos fertilized and 12 underwent biopsy. Three embryos were found to be antihuman platelet antigen-1b/1b homozygotes and two were transferred. This cycle resulted in an uneventful pregnancy and birth of a healthy child. Conclusion In cases in which there is antihuman platelet antigen incompatibility and IVIG cannot be administered, preimplantation genetic diagnosis is a reliable alternative to enable birth of unaffected children.

Background & AimsRAC1 is a guanosine triphosphatase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases are associated with dysregulation of immune defenses. We studied the role of RAC1 in inflammatory bowel diseases using human genetic and functional studies and animal models of colitis.MethodsWe used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 messenger RNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulfate sodium.ResultsWe observed a genetic association between RAC1 with ulcerative colitis in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the single nucleotide polymorphisms rs10951982 (Pcombined UC = 3.3 × 10−8, odds ratio = 1.43 [95% confidence interval: 1.26–1.63]) and rs4720672 (Pcombined UC = 4.7 × 10−6, odds ratio = 1.36 [95% confidence interval: 1.19–1.58]). Patients with inflammatory bowel disease who had the rs10951982 risk allele had increased expression of RAC1 compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected against dextran sulfate sodium–induced colitis.ConclusionsHuman studies and knockout mice demonstrated a role for the guanosine triphosphatase RAC1 in the development of ulcerative colitis; increased expression of RAC1 was associated with susceptibility to colitis.

Objectives Preimplantation genetic diagnosis (PGD) enables the identification of affected embryos prior to implantation. We present for the first time three families in which either the oocytes or embryos obtained from female carriers of mutations in the iduronate-2-sulfatase (IDS) gene underwent PGD for mucopolysaccharidosis type II (Hunter syndrome). Furthermore, we report the first ever derivation of a Hunter's syndrome (46, XX) human stem cell line from embryos (HESC) carrying the IDS and oculocutaneus albinism type 2 mutations. Methods Combined polar body (PB) 1 and 2 or a single cell of a six- to eight-cell embryo (blastomere) was used for genetic analysis by multiplex polymerase chain reaction assay using six microsatellite polymorphic markers flanking the gene and mutation. Results One couple underwent four PB-PGD cycles, with birth of a healthy girl; the second couple with one PB-PGD cycle had healthy twins; the third couple underwent seven cycles of double PGD for Hunter and Albinism syndrome with birth of healthy twins. One novel Hunter 46, XX HESC line was established displaying typical characteristics of HESC cells. Conclusions PGD is a reliable method to prevent pregnancy of children affected with Hunter syndrome. In addition, derived HESC can be further utilized for drug testing and better understanding of the pathogenesis of this syndrome. Copyright © 2011 John Wiley & Sons, Ltd.

Background Preimplantation genetic diagnosis (PGD) is a technique that enables identification of unaffected embryos prior to in vitro fertilization (IVF) transfer in couples at risk for a Mendelian disorder. Most cases involve severe genetic diseases with neurological features and/or major malformations. We present two couples in which PGD was performed for prevention of type 1 Gaucher disease, a non-neuronopathic, non-lethal disorder. Materials and methods We developed a multiplex fluorescent PCR protocol, simultaneously amplifying the familial mutations and eight closely spaced, highly polymorphic informative microsatellite markers surrounding the gene, to be used for PGD analysis. Results Couple #1 mother was homozygous for the N370S mutation and the father carried the 84GG mutation; their first daughter receives specific Gaucher therapy. One PGD cycle resulted in seven embryos of which four had the paternal wild type allele; two were transferred resulting in a healthy baby boy born at term. Couple #2, each a carrier (N370S and R359Q), whose first-born child had died (age 5 years) of Gaucher disease, underwent 7 PGD cycles. Only one cycle resulted in a clinical pregnancy but a miscarriage was followed at 10 weeks. Conclusions PGD is an effective and accurate method for preventing Gaucher disease type I in carrier couples. Since this disease is treatable, special ethical considerations and careful selection of couples should be performed.

Development of a molecular PGD protocol for a male with an X-linked deletion in the SHOX gene region, located in the pseudoautosomal region of the X/Y chromosomes. Due to excessive recombination in this region, the deletion can be found in male offspring.We developed a 13 marker multiplex fluorescent PCR protocol: 3 markers within the deleted SHOX region, 5 flanking markers, 3 informative markers on chromosome 21 (advanced maternal age) and 2 markers for sex determination.Of four embryos, two wild type males, diploid for chromosome 21 were transferred resulting in twin boys. One embryo was an affected female and another embryo was Turner. Amniocentesis confirmed the implanted embryos were males (46XY), with no recombinations.While many X-linked disorders can be analyzed by sexing, genes located in the pseudoautosomal regions have high XY recombination rates, requiring multiple markers to enable an accurate diagnosis.

Background Single cell diagnosis for preimplantation genetic diagnosis (PGD) requires simultaneous analysis of multiple linked polymorphic markers in addition to mutation analysis in order to reduce misdiagnosis. This type of analysis requires building family haplotypes spanning at least two generations. We present three childless couples in whom the female was a de novo mutation carrier in the Duchenne Muscular Dystrophy (DMD), incontinentia pigmenti (IKBKG) or Neurofibromatosis type 2 (NF2) genes, precluding linkage prior to the PGD cycle. We constructed haplotypes based on linked polymorphic markers in these families and performed concurrent diagnosis enabling embryo transfer from the first PGD cycle. Methods Informative markers flanking the DMD, IKBKG and NF2 genes were used to construct non-linked haplotypes. Polar bodies 1 (PB1) and 2 (PB2) were biopsied and analyzed to determine allelic association between the mutation and markers in multiplex PCR reactions. Results For each family, the first PGD cycle allowed the establishment of linked haplotypes based on homozygous PB1 and PB2 analysis; however, no embryos were available for transfer. Subsequent cycles, when performed, confirmed this linkage. A mutation-free child was born to the family affected with DMD and an ongoing pregnancy (32 weeks) was achieved with the carrier of the IKBKG deletion. Conclusions PB analysis for reverse linkage in real-time coupled with the PGD cycle is a powerful tool for diagnosis and linkage between markers and de novo mutations for maternal autosomal dominant or X-linked disorders. Simultaneous amplification of multiple informative markers in conjunction with the mutation allows the building of familial haplotypes and accurate PGD analysis.

Development of an efficient and reliable PGD protocol for nonsyndromic deafness, by polar body (PB) and blastomere PGD.The GJB2/GJB6 mutations along with 12 polymorphic markers were used in PGD analysis of blastomeres or polar bodies in 14 couples for 35 cycles. Marker informativity, diagnosis rates, Allele Drop Out (ADO) rates and PB1 heterozygosity rates were assessed.Six cycles were performed by PB biopsy, 27 by blastomere and two combined cycles, resulting in delivery of three unaffected children and five ongoing pregnancies. Diagnosis rates for PB and blastomeres were similar. Only 17% PB1s were heterozygote. ADO rates of 19% were observed in both groups.We have developed a single cell multiplex PGD protocol for nonsyndromic deafness with a high efficiency of diagnosis. Most PB1 are homozygous, and similar ADO rates were observed; therefore, blastomere biopsy appears to be the method of choice for this autosomal recessive disease.

Achondroplasia, the most common form of dwarfism, is a candidate for preimplantation genetic diagnosis (PGD) because a single mutation accounts for almost all cases. Multiplex fluorescent assay including the common G380R mutation in the FGFR3 gene and eight close polymorphic markers was developed. First and second polar bodies (PB) were used for PGD analysis. An affected woman was treated with routine long-protocol ovarian stimulation and puncture. In the first PGD cycle, out of four fertilized oocytes, PB analysis revealed two mutant oocytes, one with total amplification failure of the maternal allele and one with inconclusive results. In the second PGD cycle, 14 oocytes were retrieved following a higher FSH dose and by performing oocyte retrieval and by placing the patient in the anti-Trendelenburg position using abdominal pressure to allow all follicles to be drained. Following PB analysis, two embryos containing the wild-type FGFR3 allele were transferred. This led to an uncomplicated pregnancy and delivery by Caesarean section at week 38 of a healthy boy, carrying the FGFR3 wild-type maternal allele. In conclusion, oocyte retrieval, while difficult in patients with achondroplasia, can be successfully performed. PB analysis is a reliable and sensitive method for PGD for maternal achondroplasia.

Ample research demonstrates that pathophysiological levels of the pro-inflammatory cytokine interleukin-1 (IL-1) produces detrimental effects on memory functioning. However, recent evidence suggests that IL-1 may be required for the normal physiological regulation of hippocampal-dependent memory. To substantiate the physiological role of IL-1 in learning and memory we examined the induction of IL-1 gene expression following a learning experience, and the effects of IL-1 signaling blockade, by either genetic or pharmacological manipulations, on memory functioning. We show that IL-1 gene expression is induced in the hippocampus 24h following fear-conditioning in wild type mice, but not in two mouse strains with impaired IL-1 signaling. Moreover, we report that mice with transgenic over-expression of IL-1 receptor antagonist restricted to the CNS (IL-1raTG) display impaired hippocampal-dependent and intact hippocampal-independent memory in the water maze and fear-conditioning paradigms. We further demonstrate that continuous administration of IL-1ra via osmotic minipumps during prenatal development disrupt memory performance in adult mice, suggesting that IL-1 plays a critical role not only in the formation of hippocampal-dependent memory but also in normal hippocampal development. Finally, we tested the dual role of IL-1 in memory by intracerebroventricular (ICV) administration of different doses of IL-1beta and IL-1ra following learning, providing the first systematic evidence that the involvement of IL-1 in hippocampal-dependent memory follows an inverted U-shaped pattern, i.e., a slight increase in brain IL-1 levels can improve memory, whereas any deviation from the physiological range, either by excess elevation in IL-1 levels or by blockade of IL-1 signaling, results in impaired memory.

Objective The development of a preimplantation genetic diagnosis (PGD) protocol for Alagille syndrome (AGS), a rare autosomal dominant disorder with hepatic, cardiac and ophthalmologic involvement. Methods We developed a polar body (PB)-based multiplex fluorescent PCR reaction for a female affected with AGS. The protocol included analysis of the Jagged 1 (JAG1) familial mutation and five closely linked highly polymorphic markers (D20S162, D20S901, D20S894, and D20S186). Results In two cycles of PGD 9 of ten embryos were accurately diagnosed by assessment of first and second PBs, one embryo required additional blastomere biopsy. Conclusions This protocol takes advantage of the larger window of opportunity for transfer and the increased accuracy of diagnosis afforded by the combination of PB biopsy and multiple marker analysis. Two cycles resulted in the transfer of two and three mutation-free embryos and a subsequent pregnancy as measured by the rising hCG levels. Copyright © 2007 John Wiley & Sons, Ltd.

Preimplantation genetic diagnosis (PGD) for single gene defects is described for a family in which each parent is a carrier of both Tay-Sachs (TS) and Gaucher disease (GD). A multiplex fluorescent polymerase chain reaction protocol was developed that simultaneously amplified all four familial mutations and 10 informative microsatellite markers. In one PGD cycle, seven blastomeres were analysed, reaching a conclusive diagnosis in six out of seven embryos for TS and in five out of seven embryos for GD. Of the six diagnosed embryos, one was wild type for both TS and GD, and three were wild type for GD and carriers of TS. Two remaining embryos were compound heterozygotes for TS. Two transferable embryos developed into blastocysts (wt/wt and wt GD/carrier TS) and both were transferred on day 5. This single cycle of PGD resulted in a healthy live child. Allele drop-out (ADO) was observed in three of 34 reactions, yielding an 8% ADO rate. The occurrence of ADO in single cell analysis and undetected recombination events are primary causes of misdiagnosis in PGD and emphasize the need to use multiple polymorphic markers. So far as is known, this is the first report of concomitant PGD for two frequent Ashkenazi Jewish recessive disorders.

We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

Ovarian development and maintenance are poorly understood; however, diseases that affect these processes can offer insights into the underlying mechanisms. XX female gonadal dysgenesis (XX-GD) is a rare, genetically heterogeneous disorder that is characterized by underdeveloped, dysfunctional ovaries, with subsequent lack of spontaneous pubertal development, primary amenorrhea, uterine hypoplasia, and hypergonadotropic hypogonadism. Here, we report an extended consanguineous family of Palestinian origin, in which 4 females exhibited XX-GD. Using homozygosity mapping and whole-exome sequencing, we identified a recessive missense mutation in nucleoporin-107 (NUP107, c.1339G>A, p.D447N). This mutation segregated with the XX-GD phenotype and was not present in available databases or in 150 healthy ethnically matched controls. NUP107 is a component of the nuclear pore complex, and the NUP107-associated protein SEH1 is required for oogenesis in Drosophila. In Drosophila, Nup107 knockdown in somatic gonadal cells resulted in female sterility, whereas males were fully fertile. Transgenic rescue of Drosophila females bearing the Nup107D364N mutation, which corresponds to the human NUP107 (p.D447N), resulted in almost complete sterility, with a marked reduction in progeny, morphologically aberrant eggshells, and disintegrating egg chambers, indicating defective oogenesis. These results indicate a pivotal role for NUP107 in ovarian development and suggest that nucleoporin defects may play a role in milder and more common conditions such as premature ovarian failure.