Exploring the factors affecting classification and reporting of uncertain prenatal microarray findings, using a "virtual fetus" model-a pilot study.

OBJECTIVE: Significant discrepancy exists between laboratories in classification and reporting of copy number variants (CNVs). Studies exploring factors affecting prenatal CNV management are rare. Our "virtual fetus" pilot study examines these factors. METHOD: Ten prenatally diagnosed CNVs of uncertain significance (VUS) > 1Mb, encompassing OMIM-morbid genes, inherited from healthy parents, were classified by 15 MD geneticists from laboratory, prenatal, and preimplantation genetic testing (PGT) units. Geneticists addressed factors affecting classification, obligation to report, and recommendation for invasive testing or PGT. RESULTS: CNVs were classified likely benign (10.7%), VUS (74.7%), likely pathogenic (8.7%), or pathogenic (6.0%). Classification discrepancy was higher for losses versus gains. Classifying pathogenic/likely pathogenic was more common for losses (adjusted odds ratio [aOR] 10.9, 95% CI 1.55-76.9), and geneticists specializing in gynecology (aOR 4.9, 95% CI 1.03-23.3). 84.0% of respondents would report CNVs, depending on classification and family phenotype. Invasive testing in pregnancies was recommended for 29.3% of CNVs, depending on the classification and geneticist's specialization. PGT was recommended for 32.4%, depending on classification, experience years, and family's phenotype (38.0% for patients undergoing in vitro fertilization irrespectively, 26.7% otherwise). CONCLUSION: Factors affecting CNV classification/reporting are mainly dosage, family phenotype, geneticist specialization and experience. Understanding factors from our pilot study may facilitate developing an algorithm for clinical consensus and optimal management.

An expert consensus on practical clinical recommendations and guidance for patients with classic Fabry disease.

Fabry disease is an X-linked inherited lysosomal disorder that causes accumulation of glycosphingolipids in body fluids and tissues, leading to progressive organ damage and reduced life expectancy. It can affect both males and females and can be classified into classic or later-onset phenotypes. In classic Fabry disease, α-galactosidase A (α-Gal A) activity is absent or severely reduced and disease manifestations have an early onset that can affect multiple organs. In contrast, in later-onset Fabry disease, patients have residual α-Gal A activity and clinical features are primarily confined to the heart. Individualized therapeutic goals in Fabry disease are required due to varying phenotypes and patient characteristics, and the wide spectrum of disease severity. An international group of expert physicians convened to discuss and develop practical clinical recommendations for disease- and organ-specific therapeutic goals in Fabry disease, based on expert consensus and evidence identified through a structured literature review. Biomarkers reflecting involvement of various organs in adult patients with classic Fabry disease are discussed and consensus recommendations for disease- and organ-specific therapeutic goals are provided. These consensus recommendations should support the establishment of individualized approaches to the management of patients with classic Fabry disease by considering identification, diagnosis, and initiation of disease-specific therapies before significant organ involvement, as well as routine monitoring, to reduce morbidity, optimize patient care, and improve patient health-related quality of life.

Exacerbation of mild lung disorders to lethal pulmonary hypoplasia by a noncoding hypomorphic SNV in a lung-specific enhancer in trans to the frameshifting TBX4 variant.

Variants involving TBX4 are associated with a wide variety of disorders, including pulmonary arterial hypertension, ischiocoxopodopatellar syndrome (ICPPS)/small patella syndrome (SPS), lethal lung developmental disorders (LLDDs) in neonates, heart defects, and prenatally lethal posterior amelia with pelvic and pulmonary hypoplasia syndrome. The objective of our study was to elucidate the wide variable phenotypic expressivity and incomplete penetrance in a three-generation family with a truncating variant in TBX4. In addition to exome and genome sequencing analyses, a candidate noncoding regulatory single nucleotide variant (SNV) within the lung-specific TBX4 enhancer was functionally tested using an in vitro luciferase reporter assay. A heterozygous frameshift variant c.1112dup (p.Pro372Serfs*14) in TBX4 was identified in patients with mild interstitial lung disease (1), bronchiolitis obliterans (1), recurrent pneumothorax (1), ICPPS/SPS (1), LLDD (2), and in unaffected individuals (4). In two deceased neonates with LLDD, we identified a noncoding SNV rs62069651-C located in trans to the mutated TBX4 allele that reduced the TBX4 promoter activity by 63% in the reporter assay. Our findings provide a functional evidence for the recently reported model of complex compound inheritance in which both TBX4 coding and in trans noncoding hypomorphic variants in the lung-specific enhancer of TBX4 contribute to LLDD.

Expanded clinical validation of Haploseek for comprehensive preimplantation genetic testing.

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.

Preimplantation genetic testing (PGT) for copy number variants of uncertain significance (CNV- VUS) in the genomic era: to do or not to do?

PURPOSE: To review cases of couples presented to our PGT-unit with copy number variants (CNVs) classified as variants of uncertain significance (VUS) in order to better understand their needs. METHODS: Retrospective cohort study conducted in a tertiary medical-center, 2014-2019. We reviewed files of all couples applying for genetic counseling with CNVs classified as VUS. The main outcomes measured: number of VUS findings and their description, PGT-M procedures planned and performed, IVF cycles, clinical pregnancy, and live birth rates (LBR). VUS were classified according to the American-College of Medical-Genetics and Genomics classification at time of first consultation, and updated-December 2018. RESULTS: Twenty-four couples presented with a total of 30 VUS. Twelve couples (50%) had isolated VUS and 12 (50%) had VUS diagnosed in addition to a pathogenic mutation. Initially, nine findings (30%) were defined as VUS; eight (27%) as likely benign (b-VUS); and 13 (43%) as likely pathogenic (p-VUS). PGT-M was recommended for 17/30 CNVs (56.6%), 12 (70%) of which, isolated VUS. No couple had other indications for IVF. To date, nine couples performed PGT-M for isolated VUS; LBR per-couple-55.5%. Five couples performed PGT-M for both pathogenic findings and VUS, LBR-80%. After reviewing VUS classifications, 30% remained unchanged, 20% were more severely defined, and 50% less severely defined. CONCLUSION: The genomic era enables detection of VUS whose definition is subject to change as additional information becomes available. The uncertainty of variants' clinical significance and changes in VUS definition over time complicates genetic counseling. Revised guidelines for VUS interpretation and reevaluation of patient counseling before each pregnancy must be practiced when counseling them regarding the justification of PGT-M for their diagnosed VUS.

The decision-making process, experience, and perceptions of preimplantation genetic testing (PGT) users.

PURPOSE: The decision to undergo preimplantation genetic testing (PGT) entails a variety of personal and societal variables. Although PGT technology is widely accepted and used, few studies have queried the motives and concerns of PGT users; moreover, in-depth qualitative data regarding the PGT experience is scant. METHODS: In order to explore and analyze the experience, concerns, expectations, and attitudes toward the PGT technique and its implications, semi-structured interviews were conducted in a single tertiary medical center with 43 Israeli PGT users for HLA matching and autosomal dominant, autosomal recessive, and X-linked genetic disorders. RESULTS: The primary considerations in choosing PGT were prevention of birth of a child who would suffer a terminal or chronic disease as well as abrogation of a familial genetic condition. Religion played a decisive role in accepting PGT as an antenatal option. Regarding satisfaction with the PGT experience, many interviewees highlighted the need for greater attention to be given to potential stages of failure throughout the procedure and the need for emotional support. Our clinical results regarding implantation rate and cumulative live birth rate are 38-40% and 27-30%, respectively. CONCLUSION: This survey broadens understanding of the specialized needs of women, couples, and minority groups undergoing PGT and underscores the relevance of counseling services for PGT users.

Haploseek: a 24-hour all-in-one method for preimplantation genetic diagnosis (PGD) of monogenic disease and aneuploidy.

PURPOSE: To develop an economical, user-friendly, and accurate all-in-one next-generation sequencing (NGS)-based workflow for single-cell gene variant detection combined with comprehensive chromosome screening in a 24-hour workflow protocol. METHODS: We subjected single lymphoblast cells or blastomere/blastocyst biopsies from four different families to low coverage (0.3×-1.4×) genome sequencing. We combined copy-number variant (CNV) detection and whole-genome haplotype phase prediction via Haploseek, a novel, user-friendly analysis pipeline. We validated haplotype predictions for each sample by comparing with clinical preimplantation genetic diagnosis (PGD) case results or by single-nucleotide polymorphism (SNP) microarray analysis of bulk DNA from each respective lymphoblast culture donor. CNV predictions were validated by established commercial kits for single-cell CNV prediction. RESULTS: Haplotype phasing of the single lymphoblast/embryo biopsy sequencing data was highly concordant with relevant ground truth haplotypes in all samples/biopsies from all four families. In addition, whole-genome copy-number assessments were concordant with the results of a commercial kit. CONCLUSION: Our results demonstrate the establishment of a reliable method for all-in-one molecular and chromosomal diagnosis of single cells. Important features of the Haploseek pipeline include rapid sample processing, rapid sequencing, streamlined analysis, and user-friendly reporting, so as to expedite clinical PGD implementation.

Off the street phasing (OTSP): no hassle haplotype phasing for molecular PGD applications.

PURPOSE: Pre-implantation genetic diagnosis (PGD) for molecular disorders requires the construction of parental haplotypes. Classically, haplotype resolution ("phasing") is obtained by genotyping multiple polymorphic markers in both parents and at least one additional relative. However, this process is time-consuming, and immediate family members are not always available. The recent availability of massive genomic data for many populations promises to eliminate the needs for developing family-specific assays and for recruiting additional family members. In this study, we aimed to validate population-assisted haplotype phasing for PGD. METHODS: Targeted sequencing of CFTR gene variants and ~ 1700 flanking polymorphic SNPs (± 2 Mb) was performed on 54 individuals from 12 PGD families of (a) Full Ashkenazi (FA; n = 16), (b) mixed Ashkenazi (MA; n = 23 individuals with at least one Ashkenazi and one non-Ashkenazi grandparents), or (c) non-Ashkenazi (NA; n = 15) descent. Heterozygous genotype calls in each individual were phased using various whole genome reference panels and appropriate computational models. All computationally derived haplotype predictions were benchmarked against trio-based phasing. RESULTS: Using the Ashkenazi reference panel, phasing of FA was highly accurate (99.4% ± 0.2% accuracy); phasing of MA was less accurate (95.4% ± 4.5% accuracy); and phasing of NA was predictably low (83.4% ± 6.6% accuracy). Strikingly, for founder mutation carriers, our haplotyping approach facilitated near perfect phasing accuracy (99.9% ± 0.1% and 98.2% ± 2.8% accuracy for W1282X and delF508 carriers, respectively). CONCLUSIONS: Our results demonstrate the feasibility of replacing classical haplotype phasing with population-based phasing with uncompromised accuracy.

The effect of repeated biopsy on pre-implantation genetic testing for monogenic diseases (PGT-M) treatment outcome.

PURPOSE: To study the outcome of repeated biopsy for pre-implantation genetic testing in case of failed genetic diagnosis in the first biopsy. METHODS: The study group included 81 cycles where embryos underwent re-biopsy because there were no transferable embryos after the first biopsy: in 55 cycles, the first procedure was polar body biopsy (PBs) and the second cleavage-stage (BB); in 26 cycles, the first was BB and the second trophectoderm (BLAST) biopsy. The control group included 77 cycles where embryos underwent successful genetic diagnosis following the first biopsy, matched by maternal age, egg number, genetic inheritance type, and embryonic stage at the first biopsy. We measured genetic diagnosis rate, clinical pregnancy rates (PRs), live-birth rates (LBRs), gestational age, and birth weight. RESULTS: For repeated biopsy, genetic diagnosis was received in 67/81 cycles (82.7%); at a higher rate in PB + BB than in BB + BLAST (49/55, 89.1% and 18/26, 69.2% respectively, p = 0.055). Transferable embryos were found in 47 and 68 cycles in the study and the control groups. PRs/ET were 20/47 (42.6%) and 36/68 (52.9%) (p = 0.27), 16/36 (44.4%) following PB + BB, and 4/11 (36.4%) following BB + BLAST (p = 0.74). LBRs/ET were 13/47 (27.7%) in study group, and 28/68 (41.2%) in the controls (p = 0.14), 10/36 (27.8%) following PB + BB group, and 3/11 (27.3%) following BB + BLAST (p > 0.99). Gestational age and birth weight were similar in all groups. CONCLUSIONS: Re-biopsy of embryos when no genetic diagnosis could be reached following the first biopsy, achieved high rates of genetic diagnosis, pregnancies, and live births.

Children with type 1 Gaucher disease: Changing profiles in the 21st century.

Gaucher disease (GD) has phenotypic variability. Increased GD awareness especially among at-risk Ashkenazi Jews (AJ) and availability of non-invasive diagnosis induced trend to prenatal screening. We retrospectively assessed pediatric (<16years) Israeli AJ GD patients to ascertain demographics and phenotype at presentation and over-time because many were identified by large-scale screening. 55/67 patients born since 01/01/2000 are AJ with non-neuronopathic GD: 28 (50.9%) are N370S/N370S; 24 (43.6%) are N370S/other; 3 (3.5%) have no N370S allele. 30 (54.5%) diagnosed by screening; 10 (18.2%) with sibling diagnosed by screening. Of 19 (34.5%) receiving enzyme replacement therapy (ERT), 4/19 (21.1%) were by screening (N370S/N370S; N370S/L444P, N370S/84GG, N370S/IVS2+1); 15/19 (78.9%) diagnosed by symptoms and/or symptomatic sibling. 4/19 (21.1%) began ERT at age <2years; 9/19 (47.4%) at 3-5years; 6/19 (31.6%) at 6-12years. 49% presented with height/weight growth percentiles ≤25%, but group means were comparable up to 12years follow-up including 10 receiving ERT (8 for >5years). 22% presented with anemia, 20% with thrombocytopenia; at last follow-up 4% and 6%, respectively, remained cytopenic. We present a new demographic profile for pediatric GD because many identified by screening had/have few GD signs/symptoms. Nonetheless, early diagnosis is important, especially for non-N370S, non-mild genotypes.

Preimplantation genetic testing for aneuploidy by microarray analysis of polar bodies in advanced maternal age: a randomized clinical trial.

STUDY QUESTION: Does preimplantation genetic testing for aneuploidy (PGT-A) by comprehensive chromosome screening (CCS) of the first and second polar body to select embryos for transfer increase the likelihood of a live birth within 1 year in advanced maternal age women aged 36-40 years planning an ICSI cycle, compared to ICSI without chromosome analysis? SUMMARY ANSWER: PGT-A by CCS in the first and second polar body to select euploid embryos for transfer does not substantially increase the live birth rate in women aged 36-40 years. WHAT IS KNOWN ALREADY: PGT-A has been used widely to select embryos for transfer in ICSI treatment, with the aim of improving treatment effectiveness. Whether PGT-A improves ICSI outcomes and is beneficial to the patients has remained controversial. STUDY DESIGN, SIZE, DURATION: This is a multinational, multicentre, pragmatic, randomized clinical trial with intention-to-treat analysis. Of 396 women enroled between June 2012 and December 2016, 205 were allocated to CCS of the first and second polar body (study group) as part of their ICSI treatment cycle and 191 were allocated to ICSI treatment without chromosome screening (control group). Block randomization was performed stratified for centre and age group. Participants and clinicians were blinded at the time of enrolment until the day after intervention. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile couples in which the female partner was 36-40 years old and who were scheduled to undergo ICSI treatment were eligible. In those assigned to PGT-A, array comparative genomic hybridization (aCGH) analysis of the first and second polar bodies of the fertilized oocytes was performed using the 24sure array of Illumina. If in the first treatment cycle all oocytes were aneuploid, a second treatment with PB array CGH was offered. Participants in the control arm were planned for ICSI without PGT-A. Main exclusion criteria were three or more previous unsuccessful IVF or ICSI cycles, three or more clinical miscarriages, poor response or low ovarian reserve. The primary outcome was the cumulative live birth rate after fresh or frozen embryo transfer recorded over 1 year after the start of the intervention. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 205 participants in the chromosome screening group, 50 (24%) had a live birth with intervention within 1 year, compared to 45 of the 191 in the group without intervention (24%), a difference of 0.83% (95% CI: -7.60 to 9.18%). There were significantly fewer participants in the chromosome screening group with a transfer (relative risk (RR) = 0.81; 95% CI: 0.74-0.89) and fewer with a miscarriage (RR = 0.48; 95% CI: 0.26-0.90). LIMITATIONS, REASONS FOR CAUTION: The targeted sample size was not reached because of suboptimal recruitment; however, the included sample allowed a 90% power to detect the targeted increase. Cumulative outcome data were limited to 1 year. Only 11 patients out of 32 with exclusively aneuploid results underwent a second treatment cycle in the chromosome screening group. WIDER IMPLICATIONS OF THE FINDINGS: The observation that the similarity in birth rates was achieved with fewer transfers, less cryopreservation and fewer miscarriages points to a clinical benefit of PGT-A, and this form of embryo selection may, therefore, be considered to minimize the number of interventions while producing comparable outcomes. Whether these benefits outweigh drawbacks such as the cost for the patient, the higher workload for the IVF lab and the potential effect on the children born after prolonged culture and/or cryopreservation remains to be shown. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the European Society of Human Reproduction and Embryology. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. M.B.'s institution (UZBrussel) has received educational grants from IBSA, Ferring, Organon, Schering-Plough, Merck and Merck Belgium. M.B. has received consultancy and speakers' fees from Organon, Serono Symposia and Merck. G.G. has received personal fees and non-financial support from MSD, Ferring, Merck-Serono, Finox, TEVA, IBSA, Glycotope, Abbott and Gedeon-Richter as well as personal fees from VitroLife, NMC Healthcare, ReprodWissen, BioSilu and ZIVA. W.V., C.S., P.M.B., V.G., G.A., M.D., T.E.G., L.G., G.Ka., G.Ko., J.L., M.C.M., M.P., A.S., M.T., K.V., J.G. and K.S. declare no conflict of interest. TRIAL REGISTRATION NUMBER: NCT01532284. TRIAL REGISTRATION DATE: 7 February 2012. DATE OF FIRST PATIENT'S ENROLMENT: 25 June 2012.

Improvement in bone marrow infiltration in patients with type I Gaucher disease treated with taliglucerase alfa.

Preliminary data suggest a positive effect of taliglucerase alfa on the bone marrow infiltration of Gaucher cells. In this investigator-initiated study, we report the impact of taliglucerase alfa on the bone marrow fat fraction (FF) in 26 patients assessed by quantitative chemical shift imaging (QCSI). Of 15 treatment-naïve patients (median age 48 [range 24-68] years), eight had baseline FF ≤ 0.3, six of those with a FF ≤ 0.23 ('bone at risk'). All significantly improved from a median baseline FF of 0.24 (0.15-0.32) to 1st year FF of 0.37 (0.25-0.54) and 2nd year FF of 0.42 (0.27-0.59) (p = 0.01). Among the 11 'switch-over' patients (median age 42 [range 33-69] years; median imiglucerase exposure 8 [range 1-17] years), eight had baseline FF ≤ 0.3, five of those with FF < 0.23. All, but one, significantly improved from a median baseline FF of 0.17 (0.08-0.28) to 1st year FF of 0.3 (0.05-0.34) and 2nd year FF of 0.34 (0.08-0.44) (p = 0.03). Two elderly female patients (age 43 and 58 years, with 17 years imiglucerase exposure) who remained at the same enzyme replacement therapy dose, increased from baseline FF of 0.13 and 0.19 to 0.26 at 1 year. Although the number of observations is small, we hypothesize that switching to taliglucerase may result in an improved bone marrow response. A larger study is needed to assess the early benefit of taliglucerase alfa in adult patients with type 1 Gaucher disease on the bone marrow compartment.

Maternal-fetal vitamin D receptor polymorphisms significantly associated with preterm birth.

PURPOSE: Preterm birth (PTB) is a complex trait with strong genetic background, whose etiology is not fully understood. It was recently suggested that pregnancy duration is affected by fetal genetic variation even more than by the maternal genome. Vitamin D receptor (VDR) is involved in embryonic implantation and fertility. We studied the association between both maternal and neonatal vitamin D receptor (VDR) genetic variation and PTB. METHODS: Maternal and fetal (umbilical cord) DNA was isolated from Jewish Israeli idiopathic preterm newborns (24-36 weeks, n = 146) and control term newborns (>37 weeks, n = 229). Maternal and fetal VDR polymorphisms (FokI, ApaI, BsmI, TaqI) were analyzed by restriction fragment length polymorphism analysis. Using SPSS analysis to correlate VDR genotypes with phenotypic variation: pregnancy duration, preterm birth and spontaneous miscarriages, adjusted for gravidity, parity and gender of newborn. RESULTS: Women homozygous to VDR ApaI (AA) genotype had significant twofold increase risk for PTB [OR 1.973, (CI) 1.183-3.289, p = 0.009] compared to heterozygous women. Male newborns had significant (p < 0.05) 1.73-fold increase of PTB. Women with history of previous (≥1) spontaneous miscarriage had a significant increased risk for PTB if their newborn carried either of the VDR BsmI homozygous (BB or bb) genotypes compared to the heterozygous (Bb) genotype [OR 6.857, (CI) 1.273-36.934, p = 0.018 and OR 9.231, (CI) 1.753-48.618, p = 0.008, respectively], or VDR ApaI homozygous (AA or aa) genotype compared to heterozygous (Aa) genotype [OR 4.33, (CI) 1.029-18.257, p = 0.046 and OR 7.2, (CI) 1.34-38.917, p = 0.021, respectively]. CONCLUSIONS: We show association between maternal and fetal VDR genotype variants with PTB.

Prevention is the Best Therapy: The Geneticist's Approach.

Abstract During the last two decades prenatal genetic screening and diagnosis has become the cornerstone of medical care for family planning to prevent genetic disease. Carrier screening programs for genetic disorders that are prevalent in various populations identify couples and pregnancies at risk of having an affected child. These couples can proceed with a choice of invasive prenatal diagnosis tests of the fetus (chorionic villous sampling and amniocentesis), or non-invasive prenatal testing of free fetal DNA circulation in the maternal blood which has emerged within the last few years and is currently available for fetal sexing for X Linked disorders. Despite the advances in prenatal diagnosis, couples found to have a fetus affected with a genetic disorder may need to face the dilemma of pregnancy termination. Preimplantation genetic diagnosis (PGD) is an alternative to preempt risk of having a child affected with a life-altering genetic disorder. This technique allows biopsy and genetic diagnosis of embryos obtained from in vitro fertilization by analysis of the genetic material from one or a few embryonic cells. Only unaffected embryos are returned to the mother to establish the pregnancy. We present our experience using PGD for four Lysosomal storage disorders: Tay Sachs, Gaucher type 1, Hunter and Fabry disease with some of the couples being carriers of more than one genetic disorder. PGD is applicable to most disorders for which the gene and the familial mutation are known and should be presented to couples as an alternative to invasive prenatal testing.

Preimplantation genetic risk reduction: a new dilemma in the era of chromosomal microarrays and exome sequencing.

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.

A novel RYR1 pathogenic variant - Common among Libyan Jews and associated with a broad phenotypic spectrum.

Mutated skeletal muscle ryanodine receptor-1 (RYR1) gene is associated with a spectrum of autosomal dominant and recessive RyR1-related disorders with a wide phenotype. This report describes a variable phenotype associated with a previously unreported RYR1 frameshift pathogenic variant, (NM_000540.2) c.12815_12825del; p.Ala4272Glyfs*307, common in Libyan Jews. Clinical and genetic features of 14 carriers from 8 unrelated families were collected. There were 12 heterozygotes and 2 compound heterozygotes. Six heterozygotes (median age 49.8) were asymptomatic, and six (median age 24.5) presented with myopathy (n = 3) or severe arthrogryposis-like features, severe scoliosis, pes planus, post-anesthesia malignant hyperthermia, or cystic hygroma (in a fetus) (n = 1 each). None had an abnormal echocardiogram study or elevated creatine phosphokinase (CPK) levels. One bi-allelic carrier had a severe skeletal phenotype and myopathy; the other was a fetus with a cystic hygroma. Assessment of variant frequency in 447 Libyan Jews who underwent exome testing for unrelated reason yielded a prevalence of 1:55. The RYR1 p.Ala4272Glyfs*307 variant is common in Libyan Jews. It is associated with a broad phenotypic spectrum, with possible presentation among heterozygotes. Further genotype-phenotype studies are essential to delineate the clinical significance of the variant in mono- and bi-allelic carriers.

Preimplantation genetic diagnosis in genomic regions with duplications and pseudogenes: long-range PCR in the single-cell assay.

Long-range PCR is generally employed for the analysis of disease-causing mutations in genes with homologous pseudogene copies. However, long-range PCR is challenging when performed on single cells, as in preimplantation genetic diagnosis (PGD) of monogenic disorders. PGD on single cells requires concurrent analysis of a mutation together with multiple linked polymorphic markers from closely related family members to prevent misdiagnosis. In PGD cases involving childless de novo mutation carriers, linkage cannot be performed based on family members but rather must first be identified in single gametes. This can be an especially difficult task if the mutation to be assayed lies in a duplicated genomic region because gene-specific long-range PCR must be coupled with short-range PCR analysis of genetic markers on single cells. Here, we describe a novel method by which accurate PGD of pseudogene-homologous mutations can be achieved. Essentially, we performed whole genome amplification on single sperm or blastomeres followed by haplotype construction and long-range PCR-based mutation analysis. This original and universal strategy was used to establish allelic association for two different mutations in genes with one or more pseudogene copies (IKBKG and PKD1). The method was also sensitive enough to detect unexpected germline mosaicism in one mutation carrier.

Pilot study using ambroxol as a pharmacological chaperone in type 1 Gaucher disease.

The purpose of this pilot was to assess the tolerability and efficacy of ambroxol as a pharmacological chaperone in patients with symptomatic, type 1 Gaucher disease who present with measurable disease parameters but are not receiving enzyme replacement therapy (ERT) in order to provide proof of concept and/or ascertain the suitability of ambroxol for a larger clinical trial. The Israeli Ministry of Health Form 29c was employed to prescribe ambroxol for off-label use. Twelve patients were dispensed 2 capsules of 75 mg of ambroxol daily for 6 months. There were 8 females (66.7%). Mean age at entry was 41.1 (range: 24-63) years. Mean body weight at entry was 66.4 (range: 46.5-100) kg. One patient withdrew because of a hypersensitivity reaction, one because of elective splenectomy. No patient experienced clinically relevant deterioration in disease parameters measured. One patient achieved a robust response relative to baseline: +16.2% hemoglobin; +32.9% platelets; -2.8% liver volume; and -14.4% spleen volume. Three patients, including the above one, elected to continue on ambroxol for a further 6 months: hemoglobin levels and liver volumes were relatively stable, but platelet counts further increased in the above patient (+52.6% from baseline) and spleen volumes decreased further in all three patients (-6.4%, -18.6%, and -23.4% from baseline). Thus, ambroxol may be a safe option for Gaucher disease patients with potential disease-specific efficacy and should be expanded into a clinical trial using higher doses and placebo-controlled design.

Phenotype in patients with Gaucher disease and Parkinson disease.

BACKGROUND: Previous reports have shown an increased risk for Parkinson disease (PD) among type 1 Gaucher disease (GD) patients. However, the number of the reported cases of GD/PD is small and it is unknown whether certain GD phenotypes are associated with an increased PD risk. OBJECTIVE: To present GD and PD characteristics of adults affected by both diseases from a large Israeli GD clinic, and assess whether certain GD characteristics are associated with a higher risk for PD. METHODS: Medical files of patients >18years between 1990 and 2010 were reviewed for PD signs or symptoms. Available patients with PD underwent an additional neurological examination. Demographic and GD characteristics were compared between GD patients with and without PD using t-test and Fisher exact. Kaplan-Meier survival curves were used to estimate age-specific risk for PD among males and females. Age-specific risk for PD was compared between males and females using Cox Hazard ratio model. RESULTS: 510 type 1 GD adults (233 males; 45.7%), were evaluated. 11 PD patients were identified (2.2%). Among those with GD/PD cognitive impairment was common (7/11). Two patients underwent successful deep brain stimulation (DBS). PD diagnosis was associated with male gender (81.8% versus 44.9% male, p=0.027) and older age (mean age, PD=62.8, non-PD=47.1, p=0.004). GD phenotype and severity did not differ between the two groups, including mean Zimran Severity Score index (7.7 versus 8.3), percent splenectomized (15.8% versus 27.3%), history of avascular necrosis (13.0% versus 27.3%) and percent ever treated with enzyme replacement (49.4% versus 45.5%). CONCLUSION: Male gender and older age are risk factors for PD among GD patients, but GD severity is not associated with increased risk for PD. Further research is required to assess which GD patients are at a higher risk for PD, and why.

Pivotal trial with plant cell-expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease.

Taliglucerase alfa (Protalix Biotherapeutics, Carmiel, Israel) is a novel plant cell-derived recombinant human ß-glucocerebrosidase for Gaucher disease. A phase 3, double-blind, randomized, parallel-group, comparison-dose (30 vs 60 U/kg body weight/infusion) multinational clinical trial was undertaken. Institutional review board approvals were received. A 9-month, 20-infusion trial used inclusion/exclusion criteria in treatment-naive adult patients with splenomegaly and thrombocytopenia. Safety end points were drug-related adverse events: Ab formation and hypersensitivity reactions. Primary efficacy end point was reduction in splenic volume measured by magnetic resonance imaging. Secondary end points were: changes in hemoglobin, hepatic volume, and platelet counts. Exploratory parameters included biomarkers and bone imaging. Twenty-nine patients (11 centers) completed the protocol. There were no serious adverse events; drug-related adverse events were mild/moderate and transient. Two patients (6%) developed non-neutralizing IgG Abs; 2 other patients (6%) developed hypersensitivity reactions. Statistically significant spleen reduction was achieved at 9 months: 26.9% (95% confidence interval [CI]: -31.9, -21.8) in the 30-unit dose group and 38.0% (95% CI: -43.4, -32.8) in the 60-unit dose group (both P < .0001); and in all secondary efficacy end point measures, except platelet counts at the lower dose. These results support safety and efficacy of taliglucerase alfa for Gaucher disease.