A novel PKLR gene mutation identified using advanced molecular techniques

This study's purposes were to diagnose intractable hemolytic anemia and to provide guiding treatment for the affected family members. We performed NGS in a panel of 600 genes for blood diseases on a patient with obscure hemolytic anemia and her parents. We confirmed the diagnosis of pyruvate kinase deficiency, identified a novel homozygous mutation of the PKLR gene ( NM_000298 : exon 6: c.T941C: p.I314T), and ruled out other blood diseases in the Chinese family. Furthermore, amniotic fluid was taken from the mother during the second trimester, and DNA was extracted to analyze the type of PKLR gene mutation. The proband received cord blood and bone marrow from the second child of the mother for hematopoietic stem cell transplantation and achieved normal hematopoiesis. The genetic characterization analysis and genotype‐phenotype correlation study of PKLR gene suggested that NGS was an effective method to confirm the molecular diagnosis of intractable hemolytic anemia. The identification of the mutation aided in prenatal diagnosis in the second pregnancy and the effective clinical management of the affected family.     

Novel mutations in SH3TC2 in a young Japanese girl with Charcot‐Marie‐Tooth disease type 4C

Charcot‐Marie‐Tooth disease type 4C (CMT4C) is an autosomal recessive demyelinating form of CMT characterized clinically by early onset and severe spinal deformities, and is caused by mutations in SH3TC2. We describe the case of a 10‐year‐old Japanese girl diagnosed with CMT4C. The patient developed progressive foot deformities such as marked pes cavus and ankle contracture, with mild muscle weakness in both legs, and generalized areflexia. On electrophysiological studies, motor nerve conduction velocity ranged from 22.3 m/s in the tibial nerve to 48.2 m/s in the median nerve. Sensory nerve conduction velocity ranged from 30.3 m/s in the sural nerve to 52.8 m/s in the median nerve. Sequence analysis of candidate genes identified two novel heterozygous mutations, c.229C>T and c.2775G>A, in SH3TC2. The patient was diagnosed as having CMT4C with novel mutations, making this the first documented Japanese pediatric case.     

Parental Understanding of Neonatal Congenital Heart Disease

Background This study aimed to evaluate the impact of prenatal diagnosis on parental understanding of congenital heart disease (CHD) in newborns. Methods Consenting parents of newborns with CHD answered questions about the cardiac lesion, surgical repair, follow-up management, risk for CHD in future children, and maternal education before neonatal intensive care unit (NICU) discharge. A total understanding score was calculated (0–10) as the sum of five subscores: physician score, CHD score, surgery score, follow-up score, and reproduction score. Each category was scored as 0 (none correct), 1 (some correct), or 2 (all correct). The prenatal and postnatal diagnoses scores were compared. Results From June 2006 to November 2006, 50 families completed the questionnaire. Of these 50 families, 26 reported a prenatal diagnosis. The mean infant age when the parents were approached was 17.3 ± 13.3 days. The summary understanding score for the entire group was 6.3 ± 2.4 of 10. Multivariate regression analysis demonstrated a difference in scores between prenatal and postnatal diagnosis groups (p = 0.02) when control was used for maternal education. Prenatal diagnosis and maternal education (p < 0.01) had independent effects on the score. Conclusion Prenatal diagnosis increases parental understanding of neonatal CHD. Nevertheless, parental understanding remains suboptimal.     

Comprehensive and long-term outcomes of enzyme replacement therapy followed by stem cell transplantation in children with Gaucher disease type 1 and 3

Background

Gaucher disease (GD) is a lysosomal storage disorder, characterized by hepatosplenomegaly, pancytopenia, bone diseases, with or without neurological symptoms. Plasma glucosylsphingosine (lyso-Gb1), a highly sensitive and specific biomarker for GD, has been used for diagnosis and monitoring the response to treatment. Enzyme replacement therapy (ERT) is an effective treatment for the non-neurologic symptoms of GD. Neuronopathic GD (type 2 and 3) accounts for 60%–70% of the Asian affected population.

Methods

We explored combination therapy of ERT followed by hematopoietic stem cell transplantation (HSCT) and its long-term outcomes in patients with GD type 3 (GD3).

Results

Four patients with GD3 and one with GD type 1 (GD1) underwent HSCT. The types of donor were one matched-related, one matched-unrelated, and three haploidentical. The age at disease onset was 6–18 months and the age at HSCT was 3.8–15 years in the patients with GD3. The latest age at follow-up was 8–22 years, with a post-HSCT duration of 3–14 years. All patients had successful HSCT. Chronic graft-versus-host disease occurred in one patient. The enzyme activities were normalized at 2 weeks post HSCT. Lyso-Gb1 concentrations became lower than the pathological value. All of the patients are still alive and physically independent. Most of them (4/5) returned to school. None of the patients with GD3 had seizures or additional neurological symptoms after HSCT, but showed varying degrees of cognitive impairment.

Conclusions

ERT followed by HSCT could be considered as an alternative treatment for patients with GD3 who have a high risk of fatal neurological progression.     

Next‐generation sequencing of PTEN mutations for monitoring minimal residual disease in T‐cell acute lymphoblastic leukemia

Minimal residual disease (MRD) analysis has become a powerful indicator to refine therapy in acute lymphoblastic leukemia (ALL). Here, we present an MRD detection based on the next‐generation sequencing of PTEN exon 7 mutations (NGS‐PTEN) in 30 pediatric T‐cell ALL patients. By comparing the NGS‐PTEN results with current quantitative PCR of antigen receptor gene rearrangements (qPCR‐Ig/TR), an overall concordance of 80% was found between the two methods. However, the NGS‐PTEN qualified a lower number of high‐risk patients than qPCR‐Ig/TR. These findings suggest that NGS‐PTEN is a promising tool that could potentially be used to support current MRD methodologies for T‐ALL patients.     

Molecular alterations in pediatric brainstem gliomas

1 Background

Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis. Previously, diagnosis was based on a typical clinical presentation and magnetic resonance imaging findings. After the start of the era of biopsies, DIPGs bearing H3 K27 mutations have been reclassified into a novel entity, diffuse midline glioma, based on the presence of this molecular alteration. However, it is not well established how clinically diagnosed DIPG overlap with H3 K27‐mutated diffuse midline gliomas, and whether rare long‐term survivors also belong to this group.

2 Methods

We studied tumor samples obtained at diagnosis or upon autopsy from 23 children, including two long‐term survivors. Based on clinical, radiological, and histological findings, all tumors were previously diagnosed as DIPGs. All samples were analyzed for genetic alterations by next‐generation sequencing (NGS) and for protein expression by immunohistochemistry (IHC).

3 Results

H3 K27 was mutated in NGS or IHC in 20 patients, excluding both long‐term survivors. One of these long‐term survivors harbored a mutation in IDH1, formerly considered to be an alteration absent in pediatric diffuse brainstem gliomas. Other altered genes in NGS included TP53 (10 patients), MET and PDGFRA (3 patients each), VEGFR and SMARCA4 (2 patients each), and PPARγ, PTEN and EGFR in 1 patient, respectively. IHC revealed cMYC expression in 15 of 24 (63%) of all samples, exclusively in the biopsies.

4 Conclusions

Eighty‐seven percent of the tumors formerly diagnosed as DIPGs could be reclassified as H3 K27‐mutated diffuse midline gliomas. Both long‐term survivors lacked this alteration. Contrary to former conceptions, IDH1 mutations may occur also in pediatric brainstem gliomas.     

High‐dose treatment for malignant rhabdoid tumor of the kidney: No evidence for improved survival—The Gesellschaft für Pädiatrische Onkologie und Hämatologie (GPOH) experience

1 Background

Malignant rhabdoid tumor of the kidney (MRTK) is the most aggressive childhood renal tumor with overall survival (OS) rates ranging from 22% to 42%. Whether high‐dose chemotherapy with autologous stem‐cell transplantation (HDSCT) in an intensive first‐line treatment offers additional benefit is an ongoing discussion.

2 Methods

A retrospective analysis of all 58 patients with MRTK from Austria, Switzerland, and Germany treated in the framework of consecutive, prospective renal/rhabdoid tumor studies SIOP9/GPO, SIOP93‐01/GPOH (where SIOP is International Society of Pediatric Oncology and GPOH is German Society of Pediatric Oncology and Hematology), SIOP2001/GPOH, and European Rhabdoid Tumor Registry from 1991 to 2014.

3 Results

Median age at diagnosis was 11 months. Fifty percent of patients had metastases or multifocal disease at diagnosis (Stage IV). Local stage distribution was as follows: not done/I/II/III—1/6/11/40. Fifteen (26%) patients underwent upfront surgery. Thirty‐seven (64%) patients achieved a complete remission, 17 (29%) relapsed, 34 (59%) died of disease progression, and two (3%) died of treatment‐related complication. Mean time to the first event was 3.5 months. Two‐year EFS/OS (where EFS is event‐free survival) for the whole group was 37 ± 6%/38 ± 6%. Metastases/multifocal disease, younger age, and local stage III were associated with significantly inferior survival. Eleven (19%) patients underwent HDSCT (carboplatin + thiotepa, n = 6; carboplatin + etoposide + melphalan, n = 4; others, n = 1); 2‐year OS in this group was 60 ± 15% compared to 34 ± 8% in the non‐HDSCT group (P = 0.064). However, the time needed from radiologic to histologic diagnosis, stem‐cell harvest, and HDSCT must also be taken into account to avoid selection bias by excluding the highest risk group with early progression (<90 days). Thus, 2‐year EFS only for patients without progression until day 90 was 60 ± 16% consolidated by HDSCT compared to 62 ± 11% without (P = 0.8).

4 Conclusion

Our retrospective analysis suggests comparable outcomes for patients with and without HDSCT, if adjusted for early disease progression.     

Unrelated donor cord blood transplantation for non‐malignant disorders in children and adolescents

This study analyzes the data reported to the Korean Cord Blood Registry between 1994 and 2008, involving children and adolescents with non‐malignant diseases. Sixty‐five patients were evaluated in this study: SAA (n = 24), iBMFS, (n = 16), and primary immune deficiency/inherited metabolic disorder (n = 25). The CI of neutrophil recovery was 73.3% on day 42. By day 100, the CI of acute grade II–IV graft‐versus‐host disease was 32.3%. At a median follow‐up of 71 months, five‐yr OS was 50.7%. The survival rate (37.5%) and CI of neutrophil engraftment (37.5%) were lowest in patients with iBMFS. Deaths were mainly due to infection, pulmonary complications, and hemorrhage. In a multivariate analysis, the presence of >3.91 × 10⁵/kg of infused CD34 +  cells was the only factor consistently identified as significantly associated with neutrophil engraftment (p = 0.04) and OS (p = 0.03). UCBT using optimal cell doses appears to be a feasible therapy for non‐malignant diseases in children and adolescents for whom there is no appropriate HLA‐matched related donor. Strategies to reduce transplant‐related toxicities would improve the outcomes of UCBT in non‐malignant diseases.     

Diagnostic challenges for a novel SH2D1A mutation associated with X‐linked lymphoproliferative disease

Mutations in SH2D1A, encoding the intracellular adaptor signaling lymphocyte activation molecule associated protein (SAP), are associated with X‐linked lymphoproliferative disease type 1 (XLP1). We identified a novel hemizygous SH2D1A c.49G > A (p.E17K) variant in a 21‐year‐old patient with fatal Epstein‐Barr virus infection–associated hemophagocytic lymphohistiocytosis. Cellular and biochemical assays revealed normal expression of the SAP variant protein, yet binding to phosphorylated CD244 receptor was reduced by >95%. Three healthy brothers carried the SH2D1A c.49G > A variant. Thus, data suggest that this variant represents a pathogenic mutation, but with variable expressivity. Importantly, our results highlight challenges in the clinical interpretation of SH2D1A variants and caution in using functional flow cytometry assays for the diagnosis of XLP1.     

Glucosidase acid beta gene mutations in Egyptian children with Gaucher disease and relation to disease phenotypes

Introduction  

More than 200 mutations have been found in patients with Gaucher disease (GD) and some mutations usually have a high frequency in certain populations. Genotype/phenotype correlation in patients with GD has not been established. This study was designed to determine underlying mutations in Egyptian children with GD and to assess their relation to disease phenotypes.     

Molecular Characterisation and Prenatal Diagnosis of Asparto-acylase Deficiency (Canavan Disease)—Report of Two Novel and Two Known Mutations from the Indian Subcontinent

Objectives

To establish a technique for mutation identification and prenatal screening in confirmed cases of Canavan disease.

Method

Mutations in ASPA gene were identified by sequencing. Six exons of ASPA gene were amplified using intronic primers flanking the exons and then sequenced on ABI 3500Dx automated unit. This technique was used to identify mutations in three cases of Canavan disease. Prenatal diagnosis was performed in two families.

Results

Two reported mutations c.162 C?>?A (p.Asn54Lys) and c.859 G?>?A (p.Ala287Thr) were identified in two different cases of Canavan disease. Third case was compound heterozygous for two novel mutations (c.728 T?>?G, p.Ile243Ser; c.902 T?>?C, p.Leu301Pro). Prenatal diagnosis was performed in three pregnancies in two families, two affected fetuses and one unaffected fetus were identified.

Conclusions

Molecular characterization of Canavan disease helps identify the cause at genetic level, thus confirming diagnosis and enabling identification of carriers in the family. Though enzyme assay and NAA measurement allows diagnosis and prenatal diagnosis of Canavan diasease, molecular methods have the advantage of bringing accuracy in prenatal testing with an earlier result. This is the first case report of mutation studies in Canavan disease from Indian subcontinent.     

Impaired intention‐to‐treat survival after listing for liver transplantation in children with biliary atresia compared to other chronic liver diseases: 20 years’ experience from the Nordic countries

Biliary atresia (BA) is the most common indication for LT in children. We investigated whether this diagnosis per se, compared to other chronic liver diseases (OCLD), had an influence on patient survival. Data from 421 Scandinavian children, 194 with BA and 227 with OCLD, listed for LT between 1990 and 2010 were analyzed. The intention‐to‐treat survival and influencing risk factors were studied. Patients with BA had higher risk of death after listing than patients with OCLD. The youngest (<1 year) and smallest (<10 kg) children with the highest bilirubin (>510 μmol/L), highest INR (>1.6), and highest PELD score (>20) listed during 1990s had the worst outcome. Given the same PELD score, patients with BA had higher risk of death than patients with OCLD. For adolescents, low weight/BMI was the only prognostic marker. Impaired intention‐to‐treat survival in patients with BA was mainly explained by more advanced liver disease in younger ages and higher proportion of young children in the BA group rather than diagnosis per se. PELD score predicted death, but seemed to underestimate the severity of liver disease in patients with BA. Poor nutritional status and severe cholestasis had negative impact on survival, supporting the “sickest children first” allocation policy and correction of malnutrition before surgery.     

Unrelated donor hematopoietic stem cell transplantation for infantile enteropathy due to IL‐10/IL‐10 receptor defect

Recent advances in genetic diagnosis have identified mutations in gene encoding interleukin‐10 (IL‐10) and IL‐10 receptor (IL‐10R) proteins as a cause for early‐onset enterocolitis leading to hyperinflammatory immune response. Allogeneic HSCT offers a potential cure; however, it was only performed in a few infants and mainly from family‐related donors. We report a case of a girl who presented very early in life with severe infantile enterocolitis. Gene sequencing confirmed IL‐10R defect. Her older sister died at 13 months of age from severe undiagnosed enterocolitis. There was no family donor. An unrelated search identified a potential 10/10 high‐resolution HLA‐matched donor. There was some delay in donor activation because IL‐10R defect was not on the standard list of indications for unrelated HSCT. Our patient received the unrelated HSCT at seven months of age, and she is currently nine months after transplant and doing very well. Because HSCT is the curative option of choice for this disorder, we encourage adding IL‐10 and IL‐10R protein defects to the list of HSCT indications for unrelated donor procurement.     

Confidential inquiry into families with two siblings with cystic fibrosis

OBJECTIVE—To audit the care that had been provided to couples before the birth of a child with cystic fibrosis where a sibling had been previously diagnosed.
DESIGN—Retrospective review of case notes.
SAMPLE—Families where at least one affected child had been born between 1 January 1991 and 30 June 1995 and the diagnosis in the first child was made before the second affected pregnancy reached 20 weeks. The combination of information on these families with data from the prenatal diagnosis register allowed the reconstruction of a cohort of pregnancies in women with a previous affected child.
MAIN RESULTS—Forty six eligible families with a second affected child were identified. Details from the paediatrician who had diagnosed the first affected child were obtained in 43 cases: all 43 couples were offered genetic counselling, but where provided by a paediatrician this was difficult to assess as no couple was sent a summary letter. Details were obtained from the obstetrician in the subsequent affected pregnancy in 42 cases: prenatal diagnosis was not offered in 10 (24%), offered and declined in 24 (57%), offered and accepted but termination declined in eight (19%). In the overall cohort of at risk pregnancies, the estimated rate of prenatal diagnosis offer was 97%, prenatal diagnosis uptake 86%, false negative prenatal diagnosis rate 0%, and uptake of termination 95%.
CONCLUSIONS—(1) Parental choice was an important determinant of second affected births. (2) Despite widespread availability, prenatal diagnosis was not offered in an estimated 3% of at risk pregnancies. (3) There were shortcomings in counselling documentation, in particular failure to send a summary letter to counselled couples.

     

Infant feeding practices in a South African birth cohort—A longitudinal study

Childhood malnutrition is highly prevalent in low‐ and middle‐income countries. The choices of complementary foods, which are important in infant nutrition, are poorly described in this setting. We investigated infant feeding practices in a South African birth cohort, the Drakenstein Child Health Study. Longitudinal feeding data were collected from March 2012 to March 2015. Feeding practices at birth, 6–10 and 14 weeks and 6, 9, and 12 months, were investigated using food frequency questionnaires. Anthropometry was measured at birth and 12 months. The quality of the diet was analyzed using the World Health Organization infant and young child feeding indicators. Regression models were used to explore associations between feeding and growth outcomes at 1 year. Exclusive breastfeeding for 6 months was low (13%), and 19% of infants were introduced to solid foods before 4 months. There was high daily consumption of processed meat (56%) and inappropriate foods such as fruit juice (82%), soft drinks (54%), and refined sugary foods (51%) at 1 year. Dietary diversity and consumption of iron rich foods were low at 6 months (5% and 3%, respectively) but higher by 12 months (75% and 78%). Longer duration of exclusive breastfeeding was associated with a lower height‐for‐age z‐score at 1 year. Several dietary deficits and a rising trend in the consumption of inappropriate nutritionally poor foods were identified. These findings raise concern about poor dietary practices and the impact on child and long‐term health.     

Two infants with bilateral renal agenesis who were bridged by chronic peritoneal dialysis to kidney transplantation

Bilateral renal agenesis is associated with severe oligohydramnios and was considered incompatible with postnatal life due to severe pulmonary hypoplasia. The use of renal replacement therapy was limited by significant morbidity and mortality associated with dialysis in very young infants with major pulmonary pathology. In the United States, there is a tremendous controversy about whether or not the use of prenatal amniotic fluid infusions provides a benefit to fetuses with bilateral renal agenesis. One of the critical issues identified is that there are, as yet, no children reported who had achieved long‐term survival. Previous reports all indicated these children died shortly after birth or after unsuccessful peritoneal dialysis. We present two infants with a prenatal diagnosis of bilateral renal agenesis whose mothers elected to undergo prenatal amnioinfusions. One was born at 28 weeks with a birthweight of 1230 g and the other born at 34 weeks with a birthweight of 1940 g. We present the details of both cases, with initial management on chronic peritoneal dialysis, which started shortly after birth, as a bridge to living related kidney transplants.     

Early childhood development and stunting: Findings from the MAL‐ED birth cohort study in Bangladesh

Information on the association between stunting and child development is limited from low‐income settings including Bangladesh where 36% of children under‐ 5 are stunted. This study aimed to explore differences in early childhood development (ECD) between stunted (length‐for‐age z‐score [LAZ] < ?2) and nonstunted (LAZ ≥ ?2) children in Bangladesh. Children (n = 265) aged 6–24 months who participated in the MAL‐ED birth cohort study were evaluated by trained psychologists at 6, 15, and 24 months of age using the Bayley Scales of Infant and Toddler Development‐III; child length and weight were measured using standard procedures. ECD scores (z‐scores derived from cognitive, motor, language and socio‐emotional skills) were compared between stunted, underweight (weight‐for‐age z‐score < ?2), and wasted (weight‐for‐length z‐score < ?2) children, controlling for child age and sex and maternal age, education, body mass index (BMI), and depressive symptoms. Stunted children had significantly lower ECD scores than their nonstunted peers on cognitive (P = .049), motor (P < .001), language (P < .001) and social–emotional (P = .038) scales where boys had significantly lower fine motor skills compared with girls (P = .027). Mother's schooling and BMI were significant predictors of ECD. Similar to stunting, underweight children had developmental deficits in all domains (cognitive: P = .001; fine motor: P = .039, and P < .001 for both gross motor and total motor; expressive communication: P = .032; total language: P = .013; social–emotional development: P = .017). Wasted children had poor motor skills (P = .006 for the fine motor; P < .001 for both gross motor and total motor development) compared with the nonwasted peers. Early childhood stunting and underweight were associated with poor developmental outcomes in Bangladesh.     

Health utility and quality of life in pediatric liver transplant recipients

To measure HU and HRQOL in pediatric liver transplant (LT) recipients, a cross‐sectional study of patient‐parent dyads was conducted. Direct HU were assessed in 48 adolescents ≥12 years using SG and TTO techniques. Indirect HU were measured by Health Utility Index 2 and HUI3 for subjects ≥12 years and CHU9D for ≥7 years. Patients reported HRQOL using PedsQL? GC and PedsQL? TM. A total of 108 dyads participated (55.6% female; 73.2% Caucasian; 42.6% biliary atresia; 35.2% living donor; 37.0% Medicaid). Mean age at survey was 13.6 ± 3.5 years, and time from LT was 8.9 ± 4.9 years. 61.2% were on monotherapy, 25 (23.2%) had acute rejection within 3 years, and 15 (13.9%) had a biliary obstruction within 5 years. Mean indirect HU and HRQOL scores by child report were lower than norms (P < .001). LRD recipients had higher PedsQL? GC, PedsQL? TM, and HUI3 scores (P < .01). HU in pediatric LT recipients are lower than norms. Availability of HU scores for post‐transplant health states will enable measurement of quality‐adjusted life years for future comparative effectiveness studies.     

The value of 18F‐FDG PET in pediatric patients with post‐transplant lymphoproliferative disorder at initial diagnosis

PTLD is a serious complication of both solid organ and BMT. This study assessed whether ¹⁸F‐FDG PET, when added to CT scan, had additional value in the initial evaluation of PTLD in pediatric patients and whether PET/CT at baseline can reliably guide biopsy. This retrospective study evaluated 34 consecutive pediatric patients (14 female), aged 3.5–17.0 yr (mean age: 9.9 yr, s.d.: 4.9 yr), who had undergone ¹⁸F‐FDG PET/CT from May 2007 to December 2014 at initial diagnosis of PTLD following heart (n = 13), lung (n = 8), kidney (n = 4), liver (n = 3), liver and bowel (n = 3), and bone marrow (n = 3) transplantation. PTLD was diagnosed histopathologically in 33 patients and was based on clinical findings, elevated EBV, and imaging and follow‐up results in one patient. On lesion‐based analysis, ¹⁸F‐FDG PET showed more lesions than conventional CT scan (168 vs. 134), but CT revealed 22 lesions negative on PET. On per patient analysis, PET detected more lesions in 13 patients, CT identified more abnormalities in seven, and both showed the same number of lesions in 14. Adding ¹⁸F‐FDG PET to CT scans upstaged the disease in seven patients (20.5%). A combination of ¹⁸F‐FDG PET and CT was also useful in guiding biopsy, being positive in 36 of 39 samples (92.3%). These findings indicated that ¹⁸F‐FDG PET and CT are complementary at initial staging of pediatric PTLD and that ¹⁸F‐FDG PET/CT scanning can guide biopsies.