Detection of copy number variants associated with late-onset conditions in ~16 200 pregnancies: parameters for disclosure and pregnancy outcome.

BACKGROUND: Copy number variants (CNVs) associated with late-onset medical conditions are rare but important secondary findings in chromosomal microarray analysis (CMA) performed during pregnancy. Here, we critically review the cases at two tertiary centres to assess the criteria which guide the disclosure of such findings and develop a disclosure decision tool (DDT) aimed at facilitating disclosure decision. Parental decisions on receiving CNVs associated with risks for late-onset conditions were also recorded. METHODS: Prenatal CMAs in Hadassah and Shaare Zedek Medical Centers from November 2013 to October 2021 were reviewed for CNVs associated with late-onset conditions. The DDT proposed uses a five-parameter scoring system, which considers the severity, median age of onset, penetrance, understanding of genotype-phenotype correlation and actionability of the finding. RESULTS: Out of 16 238 prenatal CMAs, 16 (0.1%) harboured CNVs associated with late-onset conditions, 15 of which were disclosed. Outcome information was available on 13 of the 16 pregnancies, all of which continued to delivery. CONCLUSIONS: Our suggested DDT will help clinicians to quantitatively weigh the variables associated with CNVs of this type and arrive at a well thought out clinical decision regarding disclosure. Although the prevalence of late-onset conditions as a major finding in the prenatal setup is low, it is expected to rise with the increasing use of non-invasive CMA testing and whole exome and genome sequencing.

Diagnostic yield of chromosomal microarray and trio whole exome sequencing in cryptogenic cerebral palsy.

OBJECTIVE: To 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). METHODS: Trio 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. RESULTS: Given 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. CONCLUSIONS: Cryptogenic 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.

Combining cytogenetic and genomic technologies for deciphering challenging complex chromosomal rearrangements.

Complex chromosomal rearrangements (CCRs), a class of structural variants (SVs) involving more than two chromosome breaks, were classically thought to be extremely rare. As advanced technologies become more available, it has become apparent that CCRs are more common than formerly thought, and are a substantial cause of genetic disorders. We attempted a novel approach for solving the mechanism of challenging CCRs, which involve repetitive sequences, by precisely identifying sequence-level changes and their order. Chromosomal microarray (CMA) and FISH analyses were used for interpretation of SVs detected by whole exome sequencing (WES). Breakpoint junctions were analyzed by Nanopore sequencing, a novel long-read whole genome sequencing tool. A large deletion identified by WES, encompassing the FOXF1 enhancer, was the cause of alveolar capillary dysplasia and respiratory insufficiency, resulting in perinatal death. CMA analysis of the newborn's mother revealed two duplications encompassing the deleted region in the proband, raising our hypothesis that the deletion resulted from the mother's CCR. Breakpoint junctions of complex SVs were determined at the nucleotide level using Nanopore long-read sequencing. According to sequencing results of breakpoint junctions, the CCR in the newborn was considered the consequence of at least one double-strand break during meiosis, and reassembly of DNA fragments by intra-chromosomal homologous recombination. Our comprehensive approach, combining cytogenetics and long-read sequencing, enabled delineation of the exact breakpoints in a challenging CCR, and proposal of a mechanism in which it arises. We suggest applying our integrative approach combining technologies for deciphering future challenging CCRs, enabling risk assessment in families.

Essential Role of BRCA2 in Ovarian Development and Function.

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.).

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.

A defect in GPI synthesis as a suggested mechanism for the role of ARV1 in intellectual disability and seizures.

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.

What have we learned from 691 prenatal chromosomal microarrays for ventricular septal defects?

INTRODUCTION: Ventricular septal defect (VSD) represents the most common type of congenital cardiac anomaly, affecting more than 1 in 300 live births. The objective of this study was to examine the incidence and nature of abnormal chromosomal microarray analysis (CMA) results in a large cohort of pregnancies with VSD. MATERIAL AND METHODS: Data acquisition was performed through the Ministry of Health computerized database. All CMA results performed due to VSD during 2013-2017 were included. The rates of clinically significant CMA results of cases with isolated and non-isolated VSD were compared with two control populations-a systematic review of 9272 pregnancies and a local cohort of 5541 fetuses with normal ultrasound. RESULTS: Overall, 691 CMA analyses performed due to a sonographic indication of VSD were detected. Of 568 pregnancies with isolated VSD, eight (1.4%) clinically significant copy number variants were detected, a nonsignificant difference compared with low risk pregnancies. Of the 123 pregnancies with non-isolated VSDs, 18 (14.6%) clinically significant CMA results were detected, a considerably increased risk compared with control pregnancies. Karyotype-detectable anomalies constituted 12 of the 18 abnormal CMA results in non-isolated VSD group (66.7%), a significantly higher proportion compared with 2 of 8 (25%) in isolated VSD cohort. CONCLUSIONS: The outcomes of our study, representing the largest number of CMA results in pregnancies with VSD, suggest that the rate of abnormal CMA findings in isolated VSD does not differ from pregnancies with normal ultrasound. This observation is true for populations undergoing routine common trisomy screening tests and early sonographic evaluation, as well as widely available non-invasive prenatal screening. Conversely, CMA analysis yields a high detection rate in pregnancies with non-isolated VSD. Our results question the recommendation to perform invasive prenatal testing for CMA in pregnancies with isolated VSD.

The yield of chromosomal microarray testing for cases of abnormal fetal head circumference.

Objectives: Chromosomal microarray analysis (CMA) is the method of choice for genetic work-up in cases of fetal malformations. We assessed the detection rate of CMA in cases of abnormal fetal head circumference (HC). Methods: The study cohort was based on 81 cases of amniocenteses performed throughout Israel for the indication of microcephaly (53) or macrocephaly (28), from January 2015 through December 2018. We retrieved data regarding the clinical background, parental HCs and work-up during the pregnancy from genetic counseling summaries and from patients' medical records. Results: There was only one likely pathogenic CMA result (1.89%): a 400-kb microdeletion at 16p13.3 detected in a case of isolated microcephaly. No pathogenic results were found in the macrocephaly group. Most fetuses with microcephaly were female (87.8%), while the majority with macrocephaly were males (86.4%). Conclusions: The results imply that CMA analysis in pregnancies with microcephaly may carry a small yield compared to other indications. Regarding macrocephaly, our cohort was too small to draw conclusions. In light of the significant gender effect on the diagnosis of abnormal HC, standardization of fetal HC charts according to fetal gender may normalize cases that were categorized outside the normal range and may increase the yield of CMA for cases of abnormal HC.

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.

Mitochondrial PITRM1 peptidase loss-of-function in childhood cerebellar atrophy.

OBJECTIVE: To identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy. METHODS: Identification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients' cells and in yeast. RESULTS: Two 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. CONCLUSION: PITRM1T931M 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.

Mutant adenosine deaminase 2 in a polyarteritis nodosa vasculopathy.

BACKGROUND: Polyarteritis nodosa is a systemic necrotizing vasculitis with a pathogenesis that is poorly understood. We identified six families with multiple cases of systemic and cutaneous polyarteritis nodosa, consistent with autosomal recessive inheritance. In most cases, onset of the disease occurred during childhood. METHODS: We carried out exome sequencing in persons from multiply affected families of Georgian Jewish or German ancestry. We performed targeted sequencing in additional family members and in unrelated affected persons, 3 of Georgian Jewish ancestry and 14 of Turkish ancestry. Mutations were assessed by testing their effect on enzymatic activity in serum specimens from patients, analysis of protein structure, expression in mammalian cells, and biophysical analysis of purified protein. RESULTS: In all the families, vasculitis was caused by recessive mutations in CECR1, the gene encoding adenosine deaminase 2 (ADA2). All the Georgian Jewish patients were homozygous for a mutation encoding a Gly47Arg substitution, the German patients were compound heterozygous for Arg169Gln and Pro251Leu mutations, and one Turkish patient was compound heterozygous for Gly47Val and Trp264Ser mutations. In the endogamous Georgian Jewish population, the Gly47Arg carrier frequency was 0.102, which is consistent with the high prevalence of disease. The other mutations either were found in only one family member or patient or were extremely rare. ADA2 activity was significantly reduced in serum specimens from patients. Expression in human embryonic kidney 293T cells revealed low amounts of mutant secreted protein. CONCLUSIONS: Recessive loss-of-function mutations of ADA2, a growth factor that is the major extracellular adenosine deaminase, can cause polyarteritis nodosa vasculopathy with highly varied clinical expression. (Funded by the Shaare Zedek Medical Center and others.).

The spinal muscular atrophy with pontocerebellar hypoplasia gene VRK1 regulates neuronal migration through an amyloid-ß precursor protein-dependent mechanism.

Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH) is an infantile SMA variant with additional manifestations, particularly severe microcephaly. We previously identified a nonsense mutation in Vaccinia-related kinase 1 (VRK1), R358X, as a cause of SMA-PCH. VRK1-R358X is a rare founder mutation in Ashkenazi Jews, and additional mutations in patients of different origins have recently been identified. VRK1 is a nuclear serine/threonine protein kinase known to play multiple roles in cellular proliferation, cell cycle regulation, and carcinogenesis. However, VRK1 was not known to have neuronal functions before its identification as a gene mutated in SMA-PCH. Here we show that VRK1-R358X homozygosity results in lack of VRK1 protein, and demonstrate a role for VRK1 in neuronal migration and neuronal stem cell proliferation. Using shRNA in utero electroporation in mice, we show that Vrk1 knockdown significantly impairs cortical neuronal migration, and affects the cell cycle of neuronal progenitors. Expression of wild-type human VRK1 rescues both proliferation and migration phenotypes. However, kinase-dead human VRK1 rescues only the migration impairment, suggesting the role of VRK1 in neuronal migration is partly noncatalytic. Furthermore, we found that VRK1 deficiency in human and mouse leads to downregulation of amyloid-ß precursor protein (APP), a known neuronal migration gene. APP overexpression rescues the phenotype caused by Vrk1 knockdown, suggesting that VRK1 affects neuronal migration through an APP-dependent mechanism.

Combined mineralocorticoid and glucocorticoid deficiency is caused by a novel founder nicotinamide nucleotide transhydrogenase mutation that alters mitochondrial morphology and increases oxidative stress.

BACKGROUND: Familial 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 RESULTS: In 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. CONCLUSIONS: This 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.

Mutations in the mitochondrial seryl-tRNA synthetase cause hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis, HUPRA syndrome.

An uncharacterized multisystemic mitochondrial cytopathy was diagnosed in two infants from consanguineous Palestinian kindred living in a single village. The most significant clinical findings were tubulopathy (hyperuricemia, metabolic alkalosis), pulmonary hypertension, and progressive renal failure in infancy (HUPRA syndrome). Analysis of the consanguineous pedigree suggested that the causative mutation is in the nuclear DNA. By using genome-wide SNP homozygosity analysis, we identified a homozygous identity-by-descent region on chromosome 19 and detected the pathogenic mutation c.1169A>G (p.Asp390Gly) in SARS2, encoding the mitochondrial seryl-tRNA synthetase. The same homozygous mutation was later identified in a third infant with HUPRA syndrome. The carrier rate of this mutation among inhabitants of this Palestinian isolate was found to be 1:15. The mature enzyme catalyzes the ligation of serine to two mitochondrial tRNA isoacceptors: tRNA(Ser)(AGY) and tRNA(Ser)(UCN). Analysis of amino acylation of the two target tRNAs, extracted from immortalized peripheral lymphocytes derived from two patients, revealed that the p.Asp390Gly mutation significantly impacts on the acylation of tRNA(Ser)(AGY) but probably not that of tRNA(Ser)(UCN). Marked decrease in the expression of the nonacylated transcript and the complete absence of the acylated tRNA(Ser)(AGY) suggest that this mutation leads to significant loss of function and that the uncharged transcripts undergo degradation.

XX ovarian dysgenesis is caused by a PSMC3IP/HOP2 mutation that abolishes coactivation of estrogen-driven transcription.

XX female gonadal dysgenesis (XX-GD) is a rare, genetically heterogeneous disorder characterized by lack of spontaneous pubertal development, primary amenorrhea, uterine hypoplasia, and hypergonadotropic hypogonadism as a result of streak gonads. Most cases are unexplained but thought to be autosomal recessive. We elucidated the genetic basis of XX-GD in a highly consanguineous Palestinian family by using homozygosity mapping and candidate-gene and whole-exome sequencing. Affected females were homozygous for a 3 bp deletion (NM_016556.2, c.600_602del) in the PSMC3IP gene, leading to deletion of a glutamic acid residue (p.Glu201del) in the highly conserved C-terminal acidic domain. Proteasome 26S subunit, ATPase, 3-Interacting Protein (PSMC3IP)/Tat Binding Protein Interacting Protein (TBPIP) is a nuclear, tissue-specific protein with multiple functions. It is critical for meiotic recombination as indicated by the known role of its yeast ortholog, Hop2. Through the C terminus (not present in yeast), PSMC3IP also coactivates ligand-driven transcription mediated by estrogen, androgen, glucocorticoid, progesterone, and thyroid nuclear receptors. In cell lines, the p.Glu201del mutation abolished PSMC3IP activation of estrogen-driven transcription. Impaired estrogenic signaling can lead to ovarian dysgenesis both by affecting the size of the follicular pool created during fetal development and by failing to counteract follicular atresia during puberty. PSMC3IP joins previous genes known to be mutated in XX-GD, the FSH receptor, and BMP15, highlighting the importance of hormonal signaling in ovarian development and maintenance and suggesting a common pathway perturbed in isolated XX-GD. By analogy to other XX-GD genes, PSMC3IP is also a candidate gene for premature ovarian failure, and its role in folliculogenesis should be further investigated.

Garrod's fourth inborn error of metabolism solved by the identification of mutations causing pentosuria.

Pentosuria is one of four conditions hypothesized by Archibald Garrod in 1908 to be inborn errors of metabolism. Mutations responsible for the other three conditions (albinism, alkaptonuria, and cystinuria) have been identified, but the mutations responsible for pentosuria remained unknown. Pentosuria, which affects almost exclusively individuals of Ashkenazi Jewish ancestry, is characterized by high levels of the pentose sugar L-xylulose in blood and urine and deficiency of the enzyme L-xylulose reductase. The condition is autosomal-recessive and completely clinically benign, but in the early and mid-20th century attracted attention because it was often confused with diabetes mellitus and inappropriately treated with insulin. Persons with pentosuria were identified from records of Margaret Lasker, who studied the condition in the 1930s to 1960s. In the DCXR gene encoding L-xylulose reductase, we identified two mutations, DCXR c.583ΔC and DCXR c.52(+1)G > A, each predicted to lead to loss of enzyme activity. Of nine unrelated living pentosuric subjects, six were homozygous for DCXR c.583ΔC, one was homozygous for DCXR c.52(+1)G > A, and two were compound heterozygous for the two mutant alleles. L-xylulose reductase was not detectable in protein lysates from subjects' cells and high levels of xylulose were detected in their sera, confirming the relationship between the DCXR genotypes and the pentosuric phenotype. The combined frequency of the two mutant DCXR alleles in 1,067 Ashkenazi Jewish controls was 0.0173, suggesting a pentosuria frequency of approximately one in 3,300 in this population. Haplotype analysis indicated that the DCXR c.52(+1)G > A mutation arose more recently than the DCXR c.583ΔC mutation.

SHaploseek is a sequencing-only, high-resolution method for comprehensive preimplantation genetic testing.

Recent advances in genomic technologies expand the scope and efficiency of preimplantation genetic testing (PGT). We previously developed Haploseek, a clinically-validated, variant-agnostic comprehensive PGT solution. Haploseek is based on microarray genotyping of the embryo's parents and relatives, combined with low-pass sequencing of the embryos. Here, to increase throughput and versatility, we aimed to develop a sequencing-only implementation of Haploseek. Accordingly, we developed SHaploseek, a universal PGT method to determine genome-wide haplotypes of each embryo based on low-pass (≤ 5x) sequencing of the parents and relative(s) along with ultra-low-pass (0.2-0.4x) sequencing of the embryos. We used SHaploseek to analyze five single lymphoblast cells and 31 embryos. We validated the genome-wide haplotype predictions against either bulk DNA, Haploseek, or, at focal genomic sites, PCR-based PGT results. SHaploseek achieved > 99% concordance with bulk DNA in two families from which single cells were derived from grown-up children. In embryos from 12 PGT families, all of SHaploseek's focal site haplotype predictions were concordant with clinical PCR-based PGT results. Genome-wide, there was > 99% median concordance between Haploseek and SHaploseek's haplotype predictions. Concordance remained high at all assayed sequencing depths ≥ 2x, as well as with only 1ng of parental DNA input. In subtelomeric regions, significantly more haplotype predictions were high-confidence in SHaploseek compared to Haploseek. In summary, SHaploseek constitutes a single-platform, accurate, and cost-effective comprehensive PGT solution.

Down syndrome acute lymphoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group.

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

A deleterious founder mutation in the BMPER gene causes diaphanospondylodysostosis (DSD).

Diaphonospondylodysostosis (DSD) is a rare, recessively inherited, lethal skeletal dysplasia, characterized by severe spinal ossification, segmentation defects, and renal cystic dysplasia with nephrogenic rests. We hereby present three affected individuals: two children and a fetus from two unrelated East Jerusalem Arab-Muslim families. Whereas most fetuses die in utero or perinatally, one of the children survived to 15 months. Homozygosity mapping in the two families demonstrated a single common 3.87 Mb region on chromosome 7, ruling out previously known spondylocostal/spondylothoracic dysostosis loci. The 15 protein coding genes in the region were prioritized, and some were sequenced. A single, novel deleterious mutation, Q104X, was detected in the bone morphogenetic protein-binding endothelial regulator protein (BMPER) gene, recently reported to be mutated in other DSD patients [Funari et al., 2010]. The novel mutation we identified is an ancestral founder allele, as evidenced by a shared 440 SNP haplotype, and its frequency in the general Arab population is estimated to be <1:123. Our findings confirm loss of BMPER function as a cause of axial versus appendicular skeletal defects, and suggest that less deleterious mutations may be involved in milder axial skeleton abnormalities.