The Deciphering Developmental Disorders Study presents independent research commissioned by the Health Innovation Challenge Fund (HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (WT098051). The views expressed in this publication are those of the author(s) and not necessarily those of the Wellcome Trust or the Department of Health. The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network.Peer reviewedPublisher PD

Constantinou, Panayiotis

D'Allesandro, Mariella

Dean, John

Deciphering Developmental Disorders Study

Hauptfleisch, Catherine

Lochhead, Paul

Samant, Shalaka

The Deciphering Developmental Disorders Study presents independent research commissioned by the Health Innovation Challenge Fund (HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (WT098051). The views expressed in this publication are those of the author(s) and not necessarily those of the Wellcome Trust or the Department of Health. The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network.Peer reviewe

Aberdeen University Research

E-Mail karger@karger.com Novel Insights from Clinical Practice  Mol Syndromol 2015;6:254–258  DOI: 10.1159/000441134  A New, Atypical Case of Cobalamin F Disorder Diagnosed by Whole Exome Sequencing  Panayiotis Constantinou a    Mariella D’Alessandro a    Paul Lochhead a    Shalaka Samant b    W. Michael Bisset c    Catherine Hauptfleisch d   John Dean a    Deciphering Developmental Disorders Study Group  a  North of Scotland Regional Genetics Service, Ashgrove House,  b  Molecular Genetics Department, University of Aberdeen,  c  Department of Paediatric Gastroenterology, Royal Aberdeen Children’s Hospital, and  d  Department of Neonatology, Aberdeen Maternity Hospital,  Foresterhill , UK  Disorders of intracellular cobalamin metabolism in-clude methylmalonic acidaemia and homocystinuria. These inborn errors of metabolism can manifest with dysmorphism, developmental delay, encephalopathy and megaloblastic anaemia. Cobalamin F (cblF) is a co-factor in the vitamin B12 metabolism pathway, encoded by the  LMBRD1 gene on chromosome 6q13 [Rutsch et al., 2009]. Deficiency is inherited in an autosomal reces-sive fashion, characteristically associated with intrauter-ine growth restriction, failure to thrive, congenital heart disease, developmental delay, macrocytic anaemia, neu-tropaenia, thrombocytopaenia, hyperhomocysteinaemia and methylmalonic aciduria [Alfadhel et al., 2011]. It is usually diagnosed through urine organic acid and plas-ma amino acid analysis. Molecular analysis of  LMBRD1 can be used to confirm the genetic basis of the disease.  Key Words  Cobalamin F disorder ·  LMBRD1 mutation  Abstract  Cobalamin F (cblF) disorder, caused by homozygous or com-pound heterozygous mutations in the  LMBRD1 gene, is a rec-ognised cause of developmental delay, pancytopaenia and failure to thrive which may present in the neonatal period. A handful of cases have been reported in the medical litera-ture. We report a new case, diagnosed at the age of 6 years through whole exome sequencing, with atypical features in-cluding prominent metopic suture, cleft palate, unilateral re-nal agenesis and liver abnormalities, which broaden the phenotypic spectrum.  © 2015 The Author(s)Published by S. Karger AG, Basel  Accepted: September 14, 2015  By M. Schmid  Published online: October 14, 2015  John Dean  North of Scotland Regional Genetics Service  Ground Floor, Ashgrove House  Foresterhill, Aberdeen AB25 2ZA (UK)  E-Mail john.dean   @   nhs.net  © 2015 The Author(s)Published by S. Karger AG, Basel1661–8769/15/0065–0254$0/0  www.karger.com/msy  Established Facts  • Cobalamin F disorder is caused by homozygous or compound heterozygous mutation in  LMBRD1.  • Typical presenting features include poor growth, developmental delay and macrocytic anaemia with neutropaenia and thrombocytopaenia.  Novel Insights • Cleft palate, renal agenesis, prominent metopic suture and neonatal liver dysfunction may be features of the disorder. • Whole exome sequencing is a non-invasive route to diagnosis in rare developmental disorders where neonatal clinical and biochemical findings are inconclusive. This article is licensed under the Creative Commons Attribution 4.0 International License (CC BY) (http://www.karger.com/Services/OpenAccessLicense). Usage, derivative works and distribution are permitted provided that proper credit is given to the author and the original publisher.Downloaded by: University of Aberdeen-                                139.133.148.10 - 12/7/2016 1:58:06 PM New, Atypical Case of Cobalamin F Disorder  Mol Syndromol 2015;6:254–258 DOI: 10.1159/000441134255We present a 6-year-old child with an atypical presenta-tion of cblF disorder, diagnosed by whole exome se-quencing (WES). Case Report  The proband was referred to our genetic service at the age of 1 month due to cleft palate, thrombocytopaenia and renal agenesis. The pregnancy history was notable for pregnancy-associated hy-pertension, for which the mother was managed with methyldopa. Prenatal ultrasonography demonstrated limb shortening and de-creased liquor volumes. Delivery was by emergency lower segment Caesarian section at 32 weeks’ gestation for fetal distress. APGAR scores were 8 at 1 min and 9 at 5 min after delivery. Growth pa-rameters at birth were: weight 1,100 g (1.4th centile for gestational age), length 34 cm (<0.4th centile), and head circumference 28 cm (8th centile). Physical examination revealed a prominent metopic ridge, midline cleft soft palate, low-set ears, broad nasal tip, micro-gnathia and undescended testes with a right-sided inguinal hernia ( fig. 1 ). Ophthalmological assessment revealed abnormal retinal streaking pigmentation. Imaging demonstrated thoraco-lumbar scoliosis and right renal agenesis. A small patent ductus arteriosus with mild peripheral pulmonary artery stenosis was identified, which resolved spontaneously by the age of 3 years. He had feeding difficulties and required parenteral nutrition during the first 6–8 weeks of life. Thrombocytopaenia and neutropaenia were evident, initially attributed to sepsis and treated with antibiotics. A bone marrow examination was normal apart from neutrophil matura-tion arrest at the metamyelocyte stage, which responded to granu-locyte colony stimulating factor. He also had persistent conjugated hyperbilirubinaemia. A liver biopsy at 3 months of age revealed a paucity of bile ducts, but molecular analysis of  JAG1 for suspected Alagille syndrome was normal. Biochemical investigation at 6 weeks of age revealed a non-specific aminoaciduria with a slight-ly increased plasma homocysteine, in addition to peaks of methyl-malonic acid, p-hydroxyphenyllactic acid and p-hydroxyphenyl-pyruvic acid on organic acid analysis. These findings were attrib-uted to hepatic insult and inadequate vitamin supplementation while on parenteral nutrition. A single dose of intramuscular hy-droxocobalamin was given and folate supplementation was insti-tuted by tube feeding, continuing until the age of 3 years. Repeat biochemical studies at 2 and 3 months of age demonstrated an improvement in the biochemical profile ( table 1 ). At this time, care was transferred to another team and permanent enteral feeding was commenced by percutaneous gastrostomy. As the initial blood abnormalities appeared to have resolved, further diagnostic inves-tigation was not pursued at this stage. Severe global developmental delay was apparent on follow-up. Unaided sitting was first recorded at around 3 years of age, and walking, with a wide-based gait, was first recorded at around 6 years of age. A mild intention tremor was noted from the age of 4 years. There was no speech on the last review at age 10, but he also had bilateral sensorineural deafness and communicated using some simple Makaton signing. He had proportional short stature with a height of 116 cm (<0.4th centile), weight 23.8 kg (5th cen-tile), and a head circumference of 49 cm (<0.4th centile). Array comparative genomic hybridisation analysis (Nimblegen CGX array v1.0) was normal as was a standard karyotype. At 6 years of age, he was entered into the Deciphering Developmental Disor-ders Study and WES was performed [methods described in the  Deciphering Developmental Disorders Study, 2015]. This dem-onstrated a homozygous pathogenic mutation c.1056delG; p.Leu352fs * 18 in the  LMBRD1 gene, which was subsequently con-firmed by Sanger sequencing. Heterozygosity for this mutation was confirmed in both parents. No other potentially pathogenic vari-ants were detected in any known development gene, no regional homozygosity was detected in the exome data using BCFtools v1.0, and no evidence of isodisomy was seen. After establishing the mo-lecular diagnosis, biochemical studies were performed, revealing modestly elevated plasma homocystine and urine methylmalonic acid levels. Regular intramuscular hydroxocobalamin supplemen-tation was instituted at this time, resulting in normalisation of the a b c Fig. 1. Frontal view of the facial characteristics.  a Shortly after birth.  b At age 3 years.  c At age 6 years. Downloaded by: University of Aberdeen-                                139.133.148.10 - 12/7/2016 1:58:06 PM Constantinou   et al.  Mol Syndromol 2015;6:254–258 DOI: 10.1159/000441134256biochemical findings ( table 1 ). There has been no change in his de-velopmental status in the 2 years since starting therapy although growth parameters have improved slightly ( fig. 2 ).  Discussion  We present a new case of cblF disorder, presenting in an atypical manner with a prominent metopic suture, cleft palate, unilateral renal agenesis, feeding difficulties, and early liver abnormalities, which was diagnosed late by the use of WES. To date, a total of 15 cases have been re-ported in the literature [Rosenblatt et al., 1986; Shih et al., 1989; MacDonald et al., 1992; Wong et al., 1992; Wag-goner et al., 1998; Rutsch et al., 2009; Gailus et al., 2010; Miousse et al., 2011; Oladipo et al., 2011] in addition to a recent case of combined cblF/cblG disorder suggestive of digenic inheritance [Farwell Gonzalez et al., 2015]. The typical clinical phenotype of cblF disorder is failure to Table 1.  Biochemical findings in the probandAge Plasma  Urine CommentsMMA(<10 μmol/mmol creatinine)cysteine (20 – 80 μM)methionine(10 – 60 μM)homocystine (0 – 1 μM)homocysteine(0 – 20 μM)B12(200 – 700 ng/l) MMA(<10 μmol/mmol creatinine)homocystine(<1 μmol/mmol creatinine)6 w    29  530  modestly elevated plas-ma phenylalanine and tyrosine2 mo    11 170 >1,000 304 96 after supplementation3 mo    4 107 55 31  7.75 ys   28 <1 33 202 30 5 at molecular diagnosis8.1 ys 4 39 29 2 11 >2,000 16 after instituting hydroxocobalamin8.5 ys 12 40 34 <1 13  13  8.75 ys 5 34 34 1 13  12  9 ys <1 45 20 <1 14 <1 2  9.5 ys  40 19 <1 17     9.8 ys <1 42 29 1 14  12   MMA = Methylmalonic acidaemia; mo = months; w = weeks; ys = years. Values in bold print represent abnormal results..01 2 3 4 5 6 7 8 9 100–1–2–3–4–5–6–7–8Z-scoreAge (years)Commenced IM B12Z-HtZ-WTZ-OFC Fig. 2. Growth parameters in the proband before and after institution of intramuscu-lar (IM) hydroxocobalamin. Downloaded by: University of Aberdeen-                                139.133.148.10 - 12/7/2016 1:58:06 PM New, Atypical Case of Cobalamin F Disorder  Mol Syndromol 2015;6:254–258 DOI: 10.1159/000441134257thrive and feeding difficulties, developmental delay and haematological features including megaloblastic anae-mia, neutropaenia and thrombocytopaenia ( table 2 ). All but one of the previously described cases carry the c.1056delG; p.Leu352fs * 18. Of the 7 cases that are homo-zygous for this mutation, the majority displayed develop-mental delay, haematological findings and failure to thrive, though their phenotypes were otherwise variable. Hepatic involvement and prominent metopic suture or dysmorphism as found in our patient have been infre-quently described [Oladipo et al., 2011, Rutsch et al., 2009]. Cleft palate has not been described previously in cblF deficiency, but has been reported with other disor-ders of vitamin B12 and folic acid metabolism [Natsume et al., 1998; Weingärtner et al., 2007; Boyles et al., 2008]. Renal tubulointerstitial dysfunction has been described in disorders of cobalamin metabolism [Morath et al., 2013] as has reduced renal growth [Kruszka et al., 2013]. Renal agenesis has, however, not been described. Early institution of intramuscular hydroxocobalamin corrects the biochemical abnormalities of hyperhomocys-teinaemia and methylmalonic acidaemia and appears to limit the severity of cognitive and neurological impair-ment [Alfadhel et al., 2011]. Although late institution of intramuscular hydroxocobalamin corrected the biochem-ical profile in our patient, there has unfortunately been no effect on the severity of the patient’s developmental delay, though there appears to be a trend of improvement in growth paramaters. The initial biochemical abnormalities might have guided towards the diagnosis, but were instead attributed to the effects of parenteral nutrition as they were partly corrected with modification of the regimen. In ret-rospect, the resolution of the haematological abnormali-ties also coincided with the institution of enteral nutrition and vitamin supplementation, but did not recur when vi-tamin supplementation was subsequently reduced. By vir-tue of his continued gastrostomy feeding, some vitamin supplementation has continued throughout his child-hood, and this may explain why the usual haematological and biochemical features have not re-appeared. It should be noted that both prematurity and parenteral nutrition are associated with hepatic biochemical abnormalities [Schutzman et al., 2008]. We cannot be certain how much the cblF disorder contributed to the hepatic dysfunction in this case. This study supports the utility of a genomic test-ing approach to make an early diagnosis for neonates with a complex clinical presentation, as has been previously suggested elsewhere [Saunders et al., 2012]. The first 1,133 family trios from the Deciphering De-velopmental Disorders Study have now been reported, with an overall diagnostic yield of 27% [Deciphering De-velopmental Disorders Study, 2014; Wright et al., 2015]. One of the outcomes from the initial analysis has been the broadening of the phenotypic spectrum of previously well-described monogenic developmental disorders. The importance of high-quality or deep clinical phenotyping of rare diseases in the genomic era cannot be underesti-mated. It will improve our understanding of the natural history of these disorders, and may reveal findings that will affect clinical management. As in this case, it may re-veal new genotype-phenotype correlations, giving a bet-ter understanding of the molecular bases of developmen-tal disorders and hopefully facilitating the development of targeted therapies. In summary, this new case of cblF deficiency, diag-nosed via WES, broadens the phenotypic spectrum of the condition. It is likely that the incorporation of WES into clinical practice will highlight wider phenotypic variabil-ity in previously defined disorders.Table 2.  Clinical features of cblF disorder described in the litera-ture compared to features seen in our patientFeatures Number of affected patientsOur patientDevelopmental delay 7 +Failure to thrive 7 +Haematological features 6 +Congenital cardiac anomalies 6 +Small for gestational age 6 +Stomatitis +/– glossitis 5Gastric upset 3Dental anomalies 3Feeding difficulties 3 +Microcephaly 2 +Seizures 2Hypotonia 2Torticollis 1Pes equinovarus 1Tracheoesophageal fistula 1Intrauterine growth retardation 1 +Arthritis 1Hepatic involvement 1 +Rash 1Recurrent infection 1Facial dysmorphism 1 +Recurrent apnoea 1Encephalopathy 1Cleft palate 0 +Unilateral renal agenesis 0 +Downloaded by: University of Aberdeen-                                139.133.148.10 - 12/7/2016 1:58:06 PM Constantinou   et al.  Mol Syndromol 2015;6:254–258 DOI: 10.1159/000441134258 Acknowledgements  The Deciphering Developmental Disorders Study presents in-dependent research commissioned by the Health Innovation Challenge Fund (HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (WT098051). The views ex-pressed in this publication are those of the author(s) and not nec-essarily those of the Wellcome Trust or the Department of Health. The research team acknowledges the support of the National In-stitute for Health Research, through the Comprehensive Clinical Research Network.  Statement of Ethics  The study has UK Research Ethics Committee approval (10/H0305/83, granted by the Cambridge South REC, and GEN/284/12 granted by the Republic of Ireland REC). Disclosure Statement  The authors have no conflicts of interest to declare.  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A New, Atypical Case of Cobalamin F Disorder Diagnosed by Whole Exome Sequencing

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