Assessment of mitochondrial DNA viability ratio in day-4 biopsied embryos as an add-in to select euploid embryos for single embryo transfer.

The aim of this study was to assess mitochondrial DNA analysis as a predictor of the pregnancy potential of biopsied preimplantation embryos. The study included 78 blastomeres biopsied from day 4 cleavage stage euploid embryos. The embryo karyotype was confirmed by 24-chromosome preimplantation genetic testing for aneuploidies using the Illumina Next-Generation Sequencing (NGS) system. Mitochondria viability ratios (mtV) were determined from BAM files subjected to the web-based genome-analysis tool Galaxy. From this cohort of patients, 30.4% of patients (n = 34) failed to establish pregnancy. The mean mtV ratio [mean = 1.51 ± 1.25-1.77 (95% CI)] for this group was significantly (P < 0.01) lower compared with the embryo population that resulted in established pregnancies [mean = 2.5 ± 1.82-2.68 (95% CI)]. mtV multiple of mean (MoM) values were similarly significantly (P < 0.01) lower in blastocysts failing to establish pregnancy. At a 0.5 MoM cut-off, the sensitivity of mtV quantitation was 35.3% and specificity was 78.2%. The positive predictive value for an mtV value > 0.5 MoM was 41.4%. This study demonstrates the clinical utility of preimplantation quantification of viable mitochondrial DNA in biopsied blastomeres as a prognosticator of pregnancy potential.

Reconstruction of Tibial Plateau Fracture Malunion in the Setting of a Large Cartilage Defect in an Adolescent: A Case Report.

CASE: An 18-year-old adolescent boy presented with knee pain and stiffness secondary to tibial plateau valgus malunion and osteochondral defect, 8 months after initial injury/fixation. We opted for a novel technique that reconstructs the convex lateral tibial plateau by using osteotomy and an osteochondral autograft harvested from the lateral aspect of the ipsilateral femoral condyle. CONCLUSION: The reported novel reconstruction technique is inexpensive, achievable with routine techniques, and demonstrated a favorable short-term outcome. At 3 years of follow-up, the patient had excellent, asymptomatic, left knee mobility and function with radiographic evidence of mild posttraumatic arthritis despite normal knee alignment.

Minimally invasive percutaneous plate osteosynthesis for treatment of proximal humeral shaft fractures.

PURPOSE: The objective of this study was to evaluate the feasibility and safety of a minimally invasive percutaneous plate osteosynthesis (MIPPO) procedure for proximal humeral shaft fractures using lateral minimal proximal and distal approaches and lateral bridge plating with primary radial nerve control, and to assess its clinical and radiographic outcomes. METHODS: A retrospective review was done for the medical records of adult patients admitted for fracture of the proximal humeral shaft without associated injury to the ipsilateral upper limb and who consented to undergo a novel MIPPO technique herein reported. Patients were reviewed at regular follow-up periods and assessed at a final follow-up for evaluation of Constant, normalized Constant, and QuickDASH scores. RESULTS: There were 21 adult patients with mean age of 56 years. Three patients were lost from early follow-up; one of them had post-operative radial nerve paralysis. Eighteen patients were reviewed for the purpose of this study at a mean of 20 months of final follow-up; among them, one patient developed post-operative radial nerve paralysis with complete recovery after three months. Bone healing was achieved without any malalignment in 17 patients at a mean of 15 weeks, and one patient developed nonunion. At final assessment (mean, 20 months), the mean values of Constant, normalized Constant, and QuickDASH scores were 84 (range, 59 to 100), 95 (range, 73 to 100), and 5 (range, 0 to 18.2) respectively. CONCLUSION: Compared to pre-reported methods of MIPPO, this technique of lateral proximal and distal mini-approaches with lateral bridge plating after primary control of the radial nerve seems safe and feasible for proximal humeral shaft fractures. It gives good clinical and radiographic results with excellent restoration of upper limb function, very low incidence of post-operative radial nerve injury, and high rate of bone union in good alignment.

Role of diagnostic intracytoplasmic sperm injection (ICSI) in the management of genetically determined zona pellucida-free oocytes during in vitro fertilization: a case report.

PURPOSE: To report the utilization of diagnostic intracytoplasmic sperm injection (D-ICSI), an ICSI cycle performed in the natural cycle, to obtain information about embryo development potential after sperm injection into zona pellucida (ZP)-free oocytes. MATERIALS AND METHODS: We report the case of a couple with primary unexplained infertility with a history of previous failed, in vitro fertilization intracytoplasmic sperm injection (IVF-ICSI) cycles characterized by the presence of ZP-free oocytes. Whole exome sequencing (WES) was carried out to analyse the possible genetic basis of oocyte abnormality. RESULTS: Diagnostic ICSI provided information about the embryo development potential from ZP-free oocytes and allowed better planning of the subsequent ICSI cycle. WES revealed that the absence of ZP was likely to be due to a new (ZP1) mutation. The subsequent ICSI cycle resulted in the delivery of a healthy baby. DISCUSSION: To the best of our knowledge, our report is the first to describe the use of D-ICSI to determine the feasibility of embryo development and implantation in a patient with ZP1 mutation, resulting in the subsequent delivery of a healthy baby. We used 'diagnostic' ICSI in the normal menstrual cycle to explore the feasibility of embryo development after sperm injection into ZP-free oocytes. Our results may expand the spectrum of diagnostic procedures associated with unexplained infertility.

Partial duplication of chromosome 19 associated with syndromic duane retraction syndrome.

BACKGROUND: To evaluate possible monogenic and chromosomal anomalies in a patient with unilateral Duane retraction syndrome, modest dysmorphism, cerebral white matter abnormalities, and normal cognitive function. MATERIALS AND METHODS: Performing high-resolution array comparative genomic hybridization (array CGH) and sequencing of HOXA1, KIF21A, SALL4, and CHN1 genes. RESULTS: The proband had unilateral Duane retraction syndrome (DRS) type III on the right with low-set ears, prominent forehead, clinodactyly, and a history of frequent infections during early childhood. Motor development and cognitive function were normal. Parents were not related, and no other family member was similarly affected. MRI revealed multiple small areas of high signal on T2 weighted images in cerebral white matter oriented along white matter tracts. Sequencing of HOXA1, KIF21A, SALL4, and CHN1 did not reveal any mutation(s). Array CGH showed a 95 Kb de novo duplication on chromosome 19q13.4 encompassing four killer cell immunoglobulin-like receptor (KIR) genes. Conclusions. KIR genes have not previously been linked to a developmental syndrome, although they are known to be expressed in the human brain and brainstem and to be associated with certain infections and autoimmune diseases, including some affecting the nervous system. DRS and brain neuroimaging abnormalities may imply a central and peripheral oligodendrocyte abnormality related in some fashion to an immunomodulatory disturbance.

Morphokinetic analysis of cleavage stage embryos and its relationship to aneuploidy in a retrospective time-lapse imaging study.

PURPOSE: To analyze differences in morphokinetic parameters of chromosomally normal and aneuploid embryos utilizing time-lapse imaging and CGH microarray analysis. METHODS: This retrospective cohort study included patients undergoing IVF treatment and preimplantation genetic diagnosis for sex selection. A total of 460 embryos cultured in incubators with time-lapse imaging system (EmbryoScope) were selected for biopsy on day 3 of development. Subsequently, CGH microarray analysis was performed for aneuploidy screening of 24 chromosomes. Kinetic parameters including time for appearance of second polar body (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPBf), time to division to 2(t2), 3(t3), 4(t4), 5(t5) cells, length of second and third cell cycle (CC2= t3 t2, CC3=t5-t3), synchrony of cell division from 2 to 4 cells (S2=t4-t3) and interval t5-t2 were analyzed to compare chromosomally normal and abnormal embryos. RESULTS: The mean time durations for tPNf, t2, t5, CC2, CC3, t5-t2 differed significantly between normal and abnormal embryos. CONCLUSIONS: Time-lapse imaging morphokinetics may play a role in early prediction of aneuploid embryos due to differences in kinetic behavior that may aid in improving clinical outcome.

Nicotinic Receptor Mutation in a Mildly Dysmorphic Girl with Duane Retraction Syndrome.

BACKGROUND: To evaluate possible monogenic and chromosomal anomalies in a patient with unilateral Duane retraction syndrome and modest dysmorphism. MATERIALS AND METHODS: Clinical evaluation, sequencing of candidate genes, and array comparative genomic hybridization (array CGH). RESULTS: The proband had unilateral Duane retraction syndrome (DRS) with low-set ears bilaterally, a high arched palate, and clinodactyly. Motor development and cognitive function were normal. Parents were first cousins, but no other family member was similarly affected. No mutations were detected in the HOXA1. KIF21A. SALL4, TUBB3, and CHN1 genes. Array CGH revealed a 16 Kb de novo deletion at chromosome 8p11.2 that encompassed a portion of only one gene, the Cholinergic Receptor, Nicotinic, Beta-3 (CHRNB3, Neuronal). This gene encodes a protein that is involved in the nicotinic acetylcholine receptor on neurons. It interacts functionally with other genes that code components of the acetylcholine receptor. CONCLUSIONS: This patient's chromosomal abnormality affected only one gene that is highly expressed in the brainstem and brain, involved in neurotransmission, and could be related to her Duane retraction syndrome.

Clinical and molecular characterization of maturity onset-diabetes of the young caused by hepatocyte nuclear factor-4 alpha mutation: red flags for prediction of the diagnosis.

BACKGROUND AND OBJECTIVES: The prevalence of maturity-onset diabetes of the young (MODY) in Saudi population remains unknown, and data on molecular etiology of this condition is limited. Therefore, the present study was undertaken to elucidate clinical and molecular characteristics of a Saudi family with MODY 1. DESIGN AND SETTINGS: This is a case series study conducted at Saad Specialist Hospital in Alkhobar, Saudi Arabia. PATIENTS AND METHODS: A 12-year-old female presented to us with symptoms suggestive of diabetes. Investigations revealed hyperglycemia, glycosuria, and ketonuria without acidosis. Pancreatic antibodies were negative. She responded well to subcutaneous insulin. Her family history revealed that 2 of her siblings were diagnosed with type 1 diabetes (T1DM), while her father and mother had type 2 diabetes (T2DM). In view of this strong family history, the possibility of monogenic diabetes was raised, and the 2 genes consistent with this phenotype, hepatocyte nuclear factor-1 alpha (HNF1a) and hepatocyte nuclear factor-4 alpha (HNF4a), were studied. Accordingly, genomic DNA was isolated from peripheral blood lymphocytes of the 8 members of this family, polymerase chain reaction was carried out, and sequencing of the whole HNF4a and HNF1a genes was done. RESULTS: DNA study of the proband revealed a heterozygous substitution in intron 1 (IVS1b C > T-5)(c.50-5C > T) of the HNF1a gene. This mutation was identified in other 5 members of the family. CONCLUSION: This study alerts physicians to suspect MODY in patients who have a strongly positive family history of diabetes over a few generations with negative pancreatic antibodies and absence of ketoacidosis and obesity.

Neurologic injury in isolated sulfite oxidase deficiency.

BACKGROUND: We review clinical, neuroimaging, and genetic information on six individuals with isolated sulfite oxidase deficiency (ISOD). METHODS: All patients were examined, and clinical records, biochemistry, neuroimaging, and sulfite oxidase gene (SUOX) sequencing were reviewed. RESULTS: Data was available on six individuals from four nuclear families affected by ISOD. Each individual began to seize within the first week of life. neurologic development was arrested at brainstem reflexes, and severe microcephaly developed rapidly. neuroimaging within days of birth revealed hypoplasia of the cerebellum and corpus callosum and damage to the supratentorial brain looking like severe hypoxic-ischemic injury that evolved into cystic hemispheric white matter changes. Affected individuals all had elevated urinary S-sulfocysteine and normal urinary xanthine and hypoxanthine levels diagnostic of ISOD. Genetic studies confirmed SUOX mutations in four patients. CONCLUSIONS: ISOD impairs systemic sulfite metabolism, and yet this genetic disease affects only the brain with damage that is commonly confused with the clinical and radiologic features of severe hypoxic-ischemic encephalopathy.Lésions neurologiques dans le déficit isolé en sulfite oxydase.

Ophthalmologic observations in a patient with partial mosaic trisomy 8.

BACKGROUND: To carefully assess the phenotype and genotype of a patient with partial mosaic trisomy 8 with particular attention to ophthalmologic features. METHODS: Ophthalmologic and neuro-ophthalmologic examination; neuroimaging; conventional karyotyping; and array comparative genomic hybridization (CGH). RESULTS: The proband was the only affected child of a non-consanguineous family. At birth she was noted to have facial dysmorphism including telecanthus, low set ears, prominent nares, and an everted lower lip. She had an accommodative esotropia with otherwise normal globes, optic nerves, retinae, and orbits. She also had delayed motor milestones and mild mental retardation associated with agenesis of the corpus callosum. Both karyotyping and array CGH documented mosaic partial trisomy of chromosome 8 that included all of the "q" arm and part of the proximal "p" arm. CONCLUSIONS: This girl had a number of the classic features of mosaic trisomy 8, including an accommodative esotropia with none of the other ocular and orbital anomalies described in patients with mosaic trisomy 8. This report constitutes an initial effort to create a virtual database of patients with mosaic chromosome 8 in which careful phenotype-genotype correlation employing high resolution array CGH may help identify clues regarding the genetic etiology of ophthalmologic features of this syndrome.

Successful pregnancies after combined human leukocyte antigen direct genotyping and preimplantation genetic diagnosis utilizing multiple displacement amplification.

OBJECTIVE: To devise a new and simple technique to help select normal embryos that are human leukocyte antigen (HLA) matched to their affected siblings for diseases, such as beta-thalassemia or sickle cell anemia, which are common in this part of the world. METHODS: This study was conducted between March 2008 and April 2011 at the preimplantation Genetic Diagnosis Laboratory, Saad Specialist Hospital, Al-Khobar, Kingdom of Saudi Arabia. Embryos were obtained after 7 in vitro fertilization preimplantation genetic diagnosis (IVF-PGD) cycles. Single cells were biopsied, and extracted DNA was amplified by the multiple displacement amplification (MDA) technique. Amplified DNA was then tested for mutations in the beta-globin gene, and directly HLA typed using a sequence specific primer technique. RESULTS: We report 7 families that underwent 7 PGD cycles with HLA typing and direct HLA loci-typing using an HLA conventional commercial kit. Two patients had PGD and HLA typing for class I and II, while the other 5 had class II. The PGD cycles resulted in 3 pregnancies out of 5 patients who had HLA matched and normal embryos. One family had a successful hematopoietic stem cell transplant. CONCLUSION: This report demonstrates the first clinical application of PGD coupled with direct HLA loci-typing of DNA amplified by MDA from a single cell.

Down-regulation of OPA1 in patients with primary open angle glaucoma.

PURPOSE: Heterozygous optic atrophy type1 (OPA1) mutations are responsible for dominant optic atrophy, and the down regulation of OPA1 expression in patients with Leber hereditary optic neuropathy may imply that Opa1 protein levels in mitochondria play a role in other spontaneous optic neuropathies as well. Mitochondrial and metabolic abnormalities may put the optic nerve at risk in primary open angle glaucoma (POAG), and this preliminary study was designed to investigate whether altered OPA1 expression might be present in the progressive optic neuropathy of POAG. METHODS: Patients were eligible for inclusion if they met standard clinical criteria for POAG, including age greater than 40 years, intraocular pressure ≥ 21 mmHg in at least one eye before treatment, normal-appearing anterior chamber angles bilaterally on gonioscopy, and optic nerve injury characteristic of POAG. RNA was extracted from leukocytes and converted to cDNA by reverse transcriptase enzyme, and real time PCR was used to assess expression levels of OPA1 and the ß-globulin (HBB) housekeeping gene. The ratio of OPA1 expression to HBB expression (OPA1/HBB) for POAG patients was compared to that of controls and to clinical characteristics of POAG patients. RESULTS: Forty-three POAG patients and 27 controls were completely phenotyped with a full ophthalmologic examination and static perimetry. Mean age (POAG 67.9 years; controls 61.8 years) and sex (POAG 26 males/17 females; controls 11/16) were similar for the two groups. Mean OPA1/HBB of POAG patients (1.16, SD 0.26) was 18% lower than controls (1.41, SD 0.50), and this difference was statistically significant (p≤0.021). OPA1 expression differed between the groups (p≤0.037), but HBB expression did not differ (p≤0.24). OPA1/HBB was not correlated with any clinical feature of POAG patients. CONCLUSIONS: Transcriptional analysis of peripheral blood leucocytes is a limited model system for studying the consequences of mitochondrial abnormalities in the optic nerve. Nevertheless, OPA1 is known to affect mitochondrial stability and has now been implicated in several spontaneous optic neuropathies. Decreased OPA1 expression in POAG patients is another indication that mitochondrial function, and possibly mitochondrially-induced apoptosis, may play a role in the development of POAG.

Horizontal gaze palsy and progressive scoliosis due to a deleterious mutation in ROBO3.

PURPOSE: To describe a family with horizontal gaze palsy and progressive scoliosis with a deleterious mutation in the ROBO3 gene. METHODS: All family members had full ophthalmologic, neurologic, and orthopedic examinations and complete sequencing of the ROBO3 gene. RESULTS: Four affected members had complete loss of horizontal gaze with progressive scoliosis that varied between family members. ROBO3 sequencing revealed a novel 15 base deletion (c.2_16 delTGCTGCGCTACCTGC) in exon 1 that segregated in homozygous form with the phenotype and probably alters the shape and ionic charge of the extracellular immunoglobulin motif 1. This mutation was not detected in 100 control chromosomes. CONCLUSIONS: The novel ROBO3 mutation in this family may be among the most deleterious yet reported. Family members in general were severely affected, but comparison of this family to other families with ROBO3 mutations did not yield a definitive phenotype-genotype correlation.

High-resolution analysis of DNA copy number alterations in patients with isolated sporadic keratoconus.

PURPOSE: To determine whether patients with sporadic, non-familial keratoconus and no pathogenic mutations in the visual system homeobox 1 (VSX1) gene have evidence of chromosomal copy number alterations. METHODS: Twenty Saudi Arabian patients with isolated keratoconus, no family history of the disease and no mutations in VSX1 were recruited. Additionally, 10 ethnically-matched healthy controls were also recruited for this study. We screened patients for chromosomal copy number aberrations using the Agilent Human Genome CGH 244A Oligo Microarray Chip. RESULTS: None of the keratoconus patients screened had evidence of chromosomal copy number alterations when compared to normal ethnically matched controls. CONCLUSIONS: Chromosomal deletions and/or duplications were not detected in any of the patients tested here. Other chromosomal imbalances such as translocations, inversions, and some ploidies cannot be detected by current array CGH technology and other nuclear genetic or epigenetic factors cannot be excluded as a possible contributing factor to keratoconus pathogenesis.

Horizontal gaze palsy and progressive scoliosis without ROBO3 mutations.

BACKGROUND: To describe clinical and genetic observations in a patient with horizontal gaze palsy and progressive scoliosis (HGPPS) without identified mutations in the ROBO3 gene. MATERIALS AND METHODS: Neurologic and orthopedic evaluation of the proband; sequencing all exons, exon-intron boundaries, and promoter region of ROBO3 in the proband and his mother. Array CGH was also carried out in the proband and his mother to evaluate possible chromosomal deletion(s) and/or duplication(s). RESULTS: The proband had complete horizontal gaze restriction with full vertical gaze and small amplitude horizontal pendular nystagmus. He also had severe scoliosis and brainstem hypoplasia pathognomonic of HGPPS. However, complete sequencing of ROBO3 twice in both forward and reverse directions did not reveal any mutations. Array CGH investigation revealed no chromosomal abnormalities. CONCLUSIONS: This patient had clinical and neuroimaging characteristics considered pathognomonic of HGPPS and yet did not have ROBO3 mutations. A clinical misdiagnosis is unlikely in the absence of facial weakness (typical of Moebius syndrome), deafness (typical of the HOXA1 spectrum), or mental retardation (typical of other central decussation abnormalities). It is perhaps more likely that a phenotype identical to HGPPS can be caused by abnormalities in ROBO3 splice variant expression, by mutations of a gene other than ROBO3, or by some environmental or epigenetic factor(s) inhibiting the action of ROBO3 or its protein product in the developing brainstem.

Isolated foveal hypoplasia: report of a new case and detailed genetic investigation.

To carry out an ophthalmological and detailed genetic investigation on a 7-year-old boy with isolated foveal hypoplasia. A full ophthalmological examination and optical coherence tomography (OCT) was performed. We also performed a full genome screen for chromosomal abnormalities, and searched for mutations in two genes (GPR143 and OCA2) known to be associated with ocular albinism and PAX6 gene known to be associated with aniridia. His eye examination was normal with no iris transillumination. A fundus examination, however, showed classic signs of foveal hypoplasia. A molecular genetic investigation showed no mutation(s) in all genes screened and no chromosomal deletion(s) and/or duplication(s) were detected. We report a case of isolated foveal hypoplasia where the underlying genetic cause could not be established. We could not rule out other genetic or epigenetic factors contributing to the pathogenesis of isolated foveal hypoplasia.