Said A. Omar

Clinical Spectrum of SIX3-Associated Mutations in Holoprosencephaly: Correlation between Genotype, Phenotype, and Function.

Background: Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates. Objective: To characterise genetic and clinical findings in patients with SIX3 mutations. Methods: Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish. Results: In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%. Conclusions: Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype–phenotype correlation, as shown by functional studies using animal models.

Effects of Prenatal Steroids on Water and Sodium Homeostasis in Extremely Low Birth Weight Neonates

Objective. We sought to determine if prenatal steroid (PNS) treatment affects water and sodium (Na) balance in extremely low birth weight infants (<1000 g). Methods. PNS treatment enhances lung maturation in preterm infants and induces maturation of renal tubular function and adenylate cyclase activity in animals. We compared water and Na homeostasis for the first week of life in those infants whose mothers received steroids before delivery (PNS: n = 16) to those who did not (nonsteroid group [NSG]: n = 14). The data were collected prospectively, but PNS treatment was not given in a randomized manner. Fluids were initiated at 100 to 125 mL/kg/d and adjusted every 8 to 12 hours to allow a daily weight loss of ≤4% of birth weight and to maintain normal serum electrolytes. Weight, serum and urine electrolytes, and urine output were frequently measured and fluid intake was adjusted by increasing the amount of free water to achieve these goals. Results. When using our fluid management protocol, the percent weight loss in both groups was equivalent during each of the 7 days (15% PNS vs 17% NSG maximum loss) as well as the cumulative urine output at 1 week of age (663 mL/kg/wk PNS vs 681 mL/kg/wk NSG). PNS infants had a higher urine output on the first 2 days of life and a lower daily fluid intake for the first week. PNS infants also had significantly less insensible water loss for each of the first 4 days of life. The PNS group had a significantly lower mean peak serum Na of 138 ± 1 mmol/L vs 144 ± 2 mmol/L and none had a peak serum Na >150 mmol/L compared with 36% of the NSG infants. PNS infants had a higher cumulative Na excretion at day 2 of life (10 ± 2 mmol/kg vs 6 ± 1 mmol/kg) but a less negative cumulative Na balance at 1 week (−10 mmol/kg vs −14 mmol/kg). Conclusion. PNS treatment was associated with lower estimated insensible water loss, a decreased incidence of hypernatremia, and an earlier diuresis and natriuresis in extremely low birth weight neonates. We speculate that PNS effects these changes through enhancement of epithelial cell maturation improving skin barrier function. PNS treatment may also enhance lung Na,K-ATPase activity leading to an earlier postnatal reabsorption of fetal lung fluid increasing extracellular volume expansion to help prevent hypernatremia.

Late-Onset Neutropenia in Very Low Birth Weight Infants

Background. Neutropenia, defined as absolute neutrophil count (ANC) <1500/mm3, affects 6% to 58% of premature infants in the first week of life. This early-onset neutropenia in premature infants has previously been correlated with sepsis, maternal hypertension, severe asphyxia, and periventricular hemorrhage. Late-onset neutropenia, defined as ANC <1500/mm3 at a postnatal age of ≥3 weeks, has not been previously reported. Objectives. The purposes of this study were to determine the prevalence of late-onset neutropenia in very low birth weight (VLBW) infants and to examine the factors that may be associated with this phenomenon. Methods. A weekly complete blood cell count (CBC) was performed routinely in all premature infants with birth weight ≤1500 g (n = 225) admitted to the neonatal intensive care in a 3-year period who survived until discharge. CBC and differentials were recorded at day 1, day 3, and then weekly until discharge. The clinical data of the study infants were collected by reviewing the medical records retrospectively. Results. Late-onset neutropenia was detected in 51 infants (22%). In both neutropenic (n = 51) and nonneutropenic infants (n = 174), ANC increased postnatally, remained above 5000/mm3 for the first 3 weeks of life, and had a marked decrease at ∼4 weeks of age. Thereafter, ANC decreased to a level of ∼1400/mm3 in the neutropenic infants and 4000/mm3 in the nonneutropenic infants. The neutropenic infants had a significantly lower nadir ANC, lower hemoglobin, and higher reticulocyte count than did the nonneutropenic infants with similar platelet counts. None of the study infants received erythropoietin during their hospitalization. This late-onset neutropenia occurred at postnatal age of 6 ± 2 weeks (range: 3–10 weeks). The duration of neutropenia was 1.7 ± .7 weeks (range: 1–3 weeks). All of the neutropenic infants had anemia of prematurity with high reticulocyte count and normal platelet count. The neutropenic infants were stable, growing on full oral feedings, and had no signs or symptoms of sepsis. No adverse effects of late-onset neutropenia were apparent in these infants. Conclusion. Late-onset neutropenia is a common incidental finding in stable, growing VLBW infants that has not been previously reported. Late-onset neutropenia is a phenomenon that occurs in anemic premature infants who have marked reticulocytosis. Normal regulation of hematopoiesis is accompanied by a balance between colony-stimulating factors, such as erythropoietin and granulocyte colony-stimulating factor, which regulate erythropoiesis and granulopoiesis. We speculate that imbalance of these factors with increased reticulocytopoiesis in response to anemia of prematurity may explain this phenomenon. We recommend avoiding institution of aggressive, potentially harmful therapy for this phenomenon in healthy, growing VLBW infants.

Outcome of Extremely Low Birth Weight Infants With Leukemoid Reaction

Background. Leukemoid reaction (LR) is defined as an absolute neutrophil count (ANC) of >30 × 103/mm3. No previous study has systemically examined the clinical and prognostic significance of this phenomenon in extremely low birth weight (ELBW) infants. Objective. The purpose of this study was to examine the effect of LR in morbidity, mortality, and long-term developmental outcome in ELBW infants. Method. Infants with gestational age of ≤30 weeks and birth weight ≤1000 g were included in the study (n = 152). The medical records were reviewed for the clinical characteristics and long-term developmental outcome of these infants. Serial complete blood cell count and ANC were calculated on day 1 and weekly thereafter until discharge. LR was defined as an ANC of >30 × 103/mm3. Results. LR was detected in 17% of the study infants (26 of 152). ANC increased postnatally in LR (n = 26) and no-LR (n = 126) infants during hospitalization, peaked in the second week of life (43 ± 3 vs 14 ± 1 × 103/mm3), and remained significantly higher in LR infants during the first 5 weeks of life. LR occurred more frequently during the first 2 weeks of life and lasted for 3 ± 1 days. There was no significant difference between the LR and no-LR infants in gestational age, birth weight, delivery mode, gender, Apgar scores, or incidence of respiratory distress syndrome, patent ductus arteriosus, necrotizing enterocolitis, intraventricular hemorrhage, and retinopathy of prematurity. LR infants required a significantly longer duration of ventilatory support (36 ± 4 vs 21 ± 2 days), longer duration of oxygen requirement (58 ± 6 vs 40 ± 3 days), and had a higher incidence of bronchopulmonary dysplasia (BPD) (54% vs 25%) compared with no-LR infants. Furthermore, the length of hospitalization was significantly longer in LR infants (69 ± 6 vs 54 ± 3 days). There was no significant difference between the groups in developmental outcome at 2 years of age including receptive/expressive language, fine/gross motor skills, and hearing. Incidence of abnormal neurodevelopment outcome was also similar between LR and no-LR infants. Conclusions. LR in ELBW infants is associated with a prolonged need for ventilatory and oxygen support, a higher incidence of BPD, and a tendency for lower mortality. The findings from our study suggest that LR is associated with conditions known to have an excess of proinflammatory cytokines. Additional prospective study is needed to understand the relationship between LR, proinflammatory cytokines, and development of BPD.

Blood pressure support in extremely premature infants is affected by different courses of antenatal steroids.

Objective:  To examine the effects of partial, single and multiple courses of antenatal corticosteroids (ANS) on the need for blood pressure support in extremely premature infants.

Two cases of pontocerebellar hypoplasia: ethical and prenatal diagnostic dilemma.

We report the clinical characteristics and the outcome of two cases of pontocerebellar hypoplasia (PCH) in one family. The objective of this report is to describe the mode of presentation, discuss the clinical course, and address the dilemma of prenatal diagnosis and the prospects for genetic diagnosis for PCH. The first case is a 4-year-old boy in whom the diagnosis was made in the neonatal period. Despite extensive prenatal follow-up during the mothers subsequent pregnancy, prenatal diagnosis could not be made and a second affected child was born. Both siblings have severe developmental delay. The cases raise an important ethical dilemma about the most appropriate intervention if the mother of a child affected with PCH becomes pregnant. PCH is considered to have an autosomal-recessive mode of inheritance and a recurrence risk of 25% in each pregnancy. Until recently when genetic mutations in PCH types 2, 4, and 6 began to be identified, the lack of well-recognized genetic testing precluded experts from making clear recommendations. The best advice to these parents was difficult or elusive. With two children currently affected, should the parents terminate or continue with the latest pregnancy? Extensive monitoring with serial prenatal ultrasound failed in the previous pregnancy and resulted in the birth of the second affected child. It is evident that serial ultrasound scan may not be helpful in making the diagnosis prenatally. Therefore, other diagnostic modalities such as magnetic resonance imaging may be necessary and should be considered. With the identification of genetic basis or mutations in PCH types 2, 4, and 6 and possible development of commercial genetic testing for these types of PCH, reproductive decision or genetic testing during pregnancy should be recommended to affected families to enable informed choices.

Inhaled Prostacyclin and High-Frequency Oscillatory Ventilation in a Premature Infant With Respiratory Syncytial Virus–Associated Respiratory Failure

In a 29-day-old premature infant with respiratory syncytial virus (RSV) pneumonia, we have shown an additive effect of high-frequency oscillatory ventilation (HFOV) and continuous inhalation of prostacyclin (iPGI2) with improvement of ventilation and oxygenation. The addition of continuous inhaled iPGI2 to HFOV was beneficial in the treatment of hypoxemic respiratory failure owing to RSV-associated pneumonia. The improvement in alveolar recruitment by increasing lung expansion by HFOV along with less ventilation-perfusion mismatch by iPGI2 appears to be responsible for the synergistic effect and favorable clinical outcome. We conclude that the combined therapy of HFOV and continuous inhaled iPGI2 may be considered in RSV-associated hypoxemic respiratory failure in pediatric patients.

Expression of naive and memory T-cells in newborn infants with early-onset sepsis.

T-cells express two surface antigen isoforms that represent two different maturational stages. Naive T-cells express CD45RA and memory T-cell express CD45RO. Naive T-cells can convert to memory T-cells after antigenic exposure. We hypothesized that naive T-cell expression would be high in healthy newborns compared with adults and that memory T-cell expression would remain low in septic newborns. The expression of naive and memory T-cells was examined using flow cytometry on peripheral blood samples from 15 newborn infants (gestational age 36 ± 1 weeks) with clinical or culture positive sepsis, cord blood samples from 20 healthy newborn infants (gestational age 37 ± 1 weeks) and peripheral blood samples from 24 healthy adults. Both healthy and septic newborns demonstrated a significantly higher percentage of naive T-cells compared with adults (57 ± 2%, 67 ± 4% vs. 40 ± 2%; P P P 3 vs. 600 ± 222/mm 3 P

Genetic mutation in pontocerebellar hypoplasia

To the Editor : Pontocerebellar hypoplasias (PCH) is a heterogeneous group of rare autosomal recessive disorders characterized by a fetal onset of an abnormally small cerebellum and brainstem without significant disorganization of the brain architectures (1–5). The diagnosis of PCH is usually made clinically by features such as microcephaly, abnormal tone, and profound psychomotor delay combined with typical findings on CT scan or magnetic resonance imaging (MRI) of the brain. Neuronal degeneration (6) and maturational defects (7) in the pons, cerebellum and other brain stem nuclei may play a role in the pathogenesis of PCH. Subtype grouping of PCH based on clinical criteria is difficult. To date, six types of PCH (OMIM# 607596, 277470, 608027, 225753, 610204 and 611523) have been described based on various clinical and genetic criteria (8–10). We report the genetic etiology of PCH in two children from a consanguineous family (Figure S1, supporting information online) of Middle Eastern descent with symptoms that were not completely consistent with any of the previously described PCH types. The two patients were delivered spontaneously at term. At birth, they were found to have hypokinesis, tremulousness, hypertonia, increased upper extremity tone with tight flexion of arms, dysfunctional oral motor skills, mild retrognathia, and no obvious dysmorphism. Metabolic workup was normal. Isoelectric focusing of serum transferrin excluded congenital disorders of glycosylation. Brainstem evoked response was normal. Optic atrophy was absent. Brain CT scan and MRI showed hypoplasia of the pons and the cerebellum (11). Their anthropometric measurements stagnate at more than two standard deviations below normal in the fifth year for the older child and sixth month in the younger. Questionable visual fixation, brisk reflexes, positive Babinski, intermittent seizures and lower limb scissoring were observed in the first child. Both children suffer from profound global developmental delay. Extrapyramidal symptoms such as chorea and dystonia were yet to be appreciated in either of the two children at the ages of 4 and 1, respectively (11). Table 1 shows a summary of their clinical features. The parents of the affected children are first cousins making it probable that the disease causing allele is homozygous by descent in the affected children. We tested for homozygosity of singlenucleotide polymorphisms (SNPs) derived from candidate loci (Table S1, supporting information online), and were able to rule out three out of five causative loci; chr 7q11-21, RARS, and TSEN34, which are linked, respectively, to PCH type 3, 6, and 2 (9, 10, 12). We sequenced all exons and exon–intron junctions of TSEN2 because we found no heterozygous SNPs. This sequence was identical to the reference sequence in all members of the family. However, SNPs in the region of TSEN54 (linked to PCH type 2 and 4) showed homozygosity in the affected children, while the parents and unaffected children were heterozygous. To determine if these children harboured a mutation in TSEN54 we performed direct DNA sequence analysis of all 11 exons and the exon–intron junctions. A single point mutation, c.919G > T in exon 8 was identified. Both affected children were homozygous, while the healthy family members were heterozygous for the mutation (Fig. 1a). A diagnostic assay with

Altered thymocyte and T cell development in neonatal mice with hyperoxia-induced lung injury

Abstract Background: The adaptive immune system of neonates is relatively underdeveloped. The thymus is an essential organ for adaptive T cell development and might be affected during the natural course of oxygen induced lung injury. The effect of prolonged hyperoxia on the thymus, thymocyte and T cell development, and its proliferation has not been studied extensively. Methods: Neonatal mice were exposed to 85% oxygen (hyperoxia) or room air (normoxia) up to 28 days. Flow cytometry using surface markers were used to assay for thymocyte development and proliferation. Results: Mice exposed to prolonged hyperoxia had evidence of lung injury associated alveolar simplification, a significantly lower mean weight, smaller thymic size, lower mean thymocyte count and higher percentage of apoptotic thymocytes. T cells subpopulation in the thymus showed a significant reduction in the count and proliferation of double positive and double negative T cells. There was a significant reduction in the count and proliferation of single positive CD4+ and CD8+ T cells. Conclusions: Prolonged hyperoxia in neonatal mice adversely affected thymic size, thymocyte count and altered the distribution of T cells sub-populations. These results are consistent with the hypothesis that prolonged hyperoxia causes defective development of T cells in the thymus.