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.

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.

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.

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.

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.

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.

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.

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.

A de novo marker chromosome derived from 9p in a patient with 9p partial duplication syndrome and autism features: genotype-phenotype correlation.

BACKGROUND: Previous studies focusing on candidate genes and chromosomal regions identified several copy number variations (CNVs) associated with increased risk of autism or autism spectrum disorders (ASD). CASE PRESENTATION: We describe a 17-year-old girl with autism, severe mental retardation, epilepsy, and partial 9p duplication syndrome features in whom GTG-banded chromosome analysis revealed a female karyotype with a marker chromosome in 69% of analyzed metaphases. Array CGH analysis showed that the marker chromosome originated from 9p24.3 to 9p13.1 with a gain of 38.9 Mb. This mosaic 9p duplication was detected only in the proband and not in the parents, her four unaffected siblings, or 258 ethnic controls. Apart from the marker chromosome, no other copy number variations (CNVs) were detected in the patient or her family. Detailed analysis of the duplicated region revealed: i) an area extending from 9p22.3 to 9p22.2 that was previously identified as a critical region for the 9p duplication syndrome; ii) a region extending from 9p22.1 to 9p13.1 that was previously reported to be duplicated in a normal individual; and iii) a potential ASD locus extending from 9p24.3 to 9p23. The ASD candidate locus contained 34 genes that may contribute to the autistic features in this patient. CONCLUSION: We identified a potential ASD locus (9p24.3 to 9p23) that may encompass gene(s) contributing to autism or ASD.

A t(5;16)(p15.32;q23.3) generating 16q23.3 --> qter duplication and 5p15.32 --> pter deletion in two siblings with mental retardation, dysmorphic features, and speech delay.

We report on two siblings (half brothers on the paternal side) with a syndrome consisting of delayed development, cardiac anomalies, chest deformity, hip rotation, metatarsus adductus, genital hypoplasia, dysmorphic face, depressed nasal bridge, mental retardation, and speech delay. All metaphases examined showed a normal karyotype in the patients, their father, and both mothers. High-resolution array CGH examination revealed a 16q (6 Mb) duplication dup(16)(16q23.3 --> 16qter) and a 5p (0.97 Mb) terminal deletion del(5)(p15.32 --> pter) in both affected boys but not their healthy siblings or parents. Interphase fluorescence in situ hybridization (FISH) confirmed both the 16q duplicated region and the 5p terminal deletion. Clinical abnormalities in the patients included thin upper lip, clinodactyly, and foot deformity, which were reported previously with duplications in 16q23.3. Pectus excavatum, hip rotation, metatarsus adductus, umbilical hernia, brachycephaly, and esotropia were not reported previously in chromosome 16q duplications but may be features that occur intermittently. The 5p deleted region has been associated previously only with speech delay, which was present in both patients. These patients display certain phenotypic characteristics not reported previously in 16q duplication and confirm 5p terminal deletion as an important chromosome anomaly for speech delay.

High-resolution analysis of DNA copy number alterations in patients with primary open-angle glaucoma.

PURPOSE: To determine whether patients with isolated primary open-angle glaucoma (POAG) have evidence of chromosomal copy number alterations. METHODS: Twenty-seven Caucasian and African-American POAG patients and 12 ethnically matched controls were carefully screened for possible glaucoma and tested for chromosomal copy number alterations using high resolution array comparative genomic hybridization. RESULTS: No POAG patient had evidence of chromosomal copy number alterations when compared to normal ethnically matched controls. Additionally, there was no evidence of somatic mosaicism in any tested POAG patient. CONCLUSIONS: Chromosomal deletions and/or duplications were not detected in POAG patients as compared to controls. Other chromosomal imbalances such as translocations, inversions, and some ploidies cannot be detected by current array comparative genomic hybridization technology, and other nuclear genetic, mitochondrial abnormalities, or epigenetic factors cannot be excluded as a possible contributing factor to POAG pathogenesis.

Saudi Arabian Y-Chromosome diversity and its relationship with nearby regions.

BACKGROUND: Human origins and migration models proposing the Horn of Africa as a prehistoric exit route to Asia have stimulated molecular genetic studies in the region using uniparental loci. However, from a Y-chromosome perspective, Saudi Arabia, the largest country of the region, has not yet been surveyed. To address this gap, a sample of 157 Saudi males was analyzed at high resolution using 67 Y-chromosome binary markers. In addition, haplotypic diversity for its most prominent J1-M267 lineage was estimated using a set of 17 Y-specific STR loci. RESULTS: Saudi Arabia differentiates from other Arabian Peninsula countries by a higher presence of J2-M172 lineages. It is significantly different from Yemen mainly due to a comparative reduction of sub-Saharan Africa E1-M123 and Levantine J1-M267 male lineages. Around 14% of the Saudi Arabia Y-chromosome pool is typical of African biogeographic ancestry, 17% arrived to the area from the East across Iran, while the remainder 69% could be considered of direct or indirect Levantine ascription. Interestingly, basal E-M96* (n = 2) and J-M304* (n = 3) lineages have been detected, for the first time, in the Arabian Peninsula. Coalescence time for the most prominent J1-M267 haplogroup in Saudi Arabia (11.6 +/- 1.9 ky) is similar to that obtained previously for Yemen (11.3 +/- 2) but significantly older that those estimated for Qatar (7.3 +/- 1.8) and UAE (6.8 +/- 1.5). CONCLUSION: The Y-chromosome genetic structure of the Arabian Peninsula seems to be mainly modulated by geography. The data confirm that this area has mainly been a recipient of gene flow from its African and Asian surrounding areas, probably mainly since the last Glacial maximum onwards. Although rare deep rooting lineages for Y-chromosome haplogroups E and J have been detected, the presence of more basal clades supportive of the southern exit route of modern humans to Eurasian, were not found.

Pregnancy after preimplantation genetic diagnosis for brachydactyly type B.

Brachydactyly type B (BDB) is an autosomal dominant disease caused by mutations in the ROR2 gene. Truncating mutations lead to the severe form of the disease, which is characterized by terminal deficiency of fingers and toes. Preimplantation genetic diagnosis (PGD) was carried out in a family suffering from severe BDB. The family was screened for mutations in exons 8 and 9 and found to harbour a known nonsense mutation (c.2265C-->A) in exon 9 of the ROR2 gene, which resulted in a premature stop-codon at residue 755. Three out of 10 linked markers tested were informative for this family and single cell work-up showed amplification efficiency in over 98% of the cells. Allele drop-out (ADO) was found in 0, 4.08 and 6.1% for D9S1803, D9S1842 and D9S280 respectively. The family underwent PGD using multiple displacement amplification, fluorescent polymerase chain reaction (informative short tandem repeat) and sequencing of exon 9. Two cells were taken from the three embryos generated in the PGD cycle and the diagnosis of both cells separately showed one normal embryo free of BDB abnormal allele. This embryo was transferred back to the mother and resulted in a singleton pregnancy. Postnatal DNA testing of the newborn confirmed the PGD result.

Five new consanguineous families with horizontal gaze palsy and progressive scoliosis and novel ROBO3 mutations.

Horizontal gaze palsy and progressive scoliosis (HGPPS) is an autosomal recessive neurologic disorder caused by homozygous or compound heterozygous mutations in the ROBO3 gene on chromosome 11. We clinically evaluated seven individuals with HGPPS from five previously unreported consanguineous families. We sequenced ROBO3 in all affected individuals, additional unaffected members of each family, and ethnic controls. All affected individuals had severe horizontal gaze restriction, progressive scoliosis, and lower brainstem hypoplasia on neuroimaging, the hallmarks of this syndrome. One individual experienced head trauma with a right subdural hematoma associated with a right hemiparesis, observations that confirm clinically for the first time that corticospinal tracts in HGPPS are uncrossed. We found five novel homozygous ROBO3 mutations (four missense mutations and one base deletion) distributed throughout the extracellular domain of the gene. The ROBO3 gene does not appear to have an obvious hot spot area for mutations; therefore, we recommend sequencing all exons and exon-intron boundaries in patients with clinical and/or radiologic features of HGPPS.

Delivery of a normal baby after preimplantation genetic diagnosis for non-ketotic hyperglycinaemia.

Non-ketotic hyperglycinaemia (NKH), or glycine encephalopathy, is an autosomal recessive neurometabolic disease caused by defective activity of the glycine cleavage system. Up to 80% of NKH cases are caused by mutations in the P protein encoded by the glycine decarboxylase (GLDC) gene. GLDC deletions were identified in approximately 20% of NKH mutant alleles and resulted in a severe neonatal form of the disease. Given the difficult management of NKH caused by GLDC deletion, it was decided to adopt a preventative approach in a family with a history of this disease by using preimplantation genetic diagnosis (PGD). In this family, there is a deletion in the 5' UTR (untranslated region) up to the third intron of GLDC. PGD was carried out using multiple displacement amplification (MDA) and fluorescent polymerase chain reaction (PCR). This resulted in a singleton pregnancy after transfer of three unaffected embryos. Post-natal DNA testing of the newborn confirmed the PGD result. This is the first report of a successful PGD cycle intended to prevent the occurrence of NKH in a family with a history of the disease. The use of MDA coupled with fluorescent PCR is a very encouraging strategy leading to both low allele drop-out (2/40) and failure of amplification (0/40) rates.

Successful pregnancies after application of array-comparative genomic hybridization in PGS-aneuploidy screening.

Recurrent IVF failure, implantation failure and early embryo demise can be attributed to the high frequency of chromosomal aneuploidy observed in human embryos. Preimplantation genetic screening (PGS) using multiple displacement amplifications (MDA) and array comparative genomic hybridization (aCGH) was successfully performed on eight patients with a minimum of seven recurrent IVF failures with the aim of detecting aneuploidy and ameliorating pregnancy rate. A total of 41 embryos with eight or more cells were biopsied by taking two blastomeres from each embryo. The DNA from these blastomeres were amplified and analysed by aCGH technology. The aCGH results showed a complex panel of chromosomal abnormalities in 60% of the diagnosed embryos. Some abnormalities could not be detected by the seven-probe panel (13, 16, 18, 21, 22, X and Y) used in fluorescence in-situ hybridization. Six out of eight patients had embryos for transfer with five out of those six showing positive pregnancy tests. As far as is known, this report is the first to show a pregnancy after PGS using the aCGH technology. The pregnancy rate obtained here is encouraging and will open the door for enrollment of more patients.