European Journal of Medical Genetics 57 (2014) 288e292
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
New clinically defined syndrome
A newly recognized syndrome of severe growth deficiency, microcephaly, intellectual disability, and characteristic facial features Chana Vinkler a, b, *, Esther Leshinsky-Silver b, c, e, Marina Michelson a, b, Dorothea Haas f, Tally Lerman-Sagie b, d, e, Dorit Lev a, b, e a
Institute of Medical Genetics, Wolfson Medical Center, Holon, Israel Metabolic Neurogenetic Service, Wolfson Medical Center, Holon, Israel Molecular Genetics Laboratory, Wolfson Medical Center, Holon, Israel d Pediatric Neurology Unit, Wolfson Medical Center, Holon, Israel e Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel f Division of Inborn Metabolic Diseases, University Children’s Hospital, Im Neuenheimer Feld, Heidelberg, Germany b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2013 Accepted 25 March 2014 Available online 5 April 2014
Genetic syndromes with proportionate severe short stature are rare. We describe two sisters born to nonconsanguineous parents with severe linear growth retardation, poor weight gain, microcephaly, characteristic facial features, cutaneous syndactyly of the toes, high myopia, and severe intellectual disability. During infancy and early childhood, the girls had transient hepatosplenomegaly and low blood cholesterol levels that normalized later. A thorough evaluation including metabolic studies, radiological, and genetic investigations were all normal. Cholesterol metabolism and transport were studied and no definitive abnormality was found. No clinical deterioration was observed and no metabolic crises were reported. After due consideration of other known hereditary causes of post-natal severe linear growth retardation, microcephaly, and intellectual disability, we propose that this condition represents a newly recognized autosomal recessive multiple congenital anomaly-intellectual disability syndrome. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Microcephaly Small stature Intellectual disability Low cholesterol
1. Introduction
2. Patients report
Genetic syndromes involving severe postnatal proportionate short stature are rare. Numerous etiologies should be considered when investigating patients who present with familial severe growth retardation and systemic involvement. As was previously discussed [Hall, 2010], these include metabolic diseases, congenital anomalies, endocrine disorders, nutritional factors, exposure to teratogens, exposure to congenital infections, chromosomal aberrations, and many genetic disorders. We present two sisters with postnatal proportionate short stature, microcephaly, high myopia, severe intellectual disability, dysmorphic features, and transient low cholesterol levels. We suggest that this is a newly recognized syndrome probably inherited in an autosomal recessive pattern.
2.1. Patient 1
* Corresponding author. Institute of Medical Genetics, Wolfson Medical Center, Holon 58100, Israel. Tel.: þ97 235028536; fax: þ97 235028566. E-mail addresses: [email protected] (C. Vinkler), [email protected] (D. Lev). http://dx.doi.org/10.1016/j.ejmg.2014.03.010 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
The patient was the first child of seven children. Five of the children (three boys and two girls) have normal growth and development. She was born to a healthy 22-year-old woman and her unrelated 23-year-old husband, both of Ashkenazi Jewish ancestry. They had no early miscarriages. The pregnancy was uneventful and she was born at term via an uncomplicated delivery. Her birth weight was 2930 g (w15th centile), her supine length was 47 cm (w5th centile) and her head circumference was 33 cm (w10th centile). Dysmorphic features were noticed at birth including short nose, depressed nasal bridge, low set ears, short neck, clinodactyly, and cutaneous syndactyly of T2-3. Mild edema of the upper thorax, hypotonia, and strabismus were also noted. Cranial ultrasound showed an intraventricular hemorrhage (grade IIeIII) and caudothalamic cysts. Thyroid function tests were normal. Two weeks later, a repeat cranial ultrasound was normal and no cysts were demonstrated. Around the age of 6 months growth retardation had been noted. Her weight was 5150 g, around 3 SD
C. Vinkler et al. / European Journal of Medical Genetics 57 (2014) 288e292
below the mean for her age, and supine length was 58 cm, around 4 SD below the mean for her age. At the age of one year, she was hospitalized due to severe failure to thrive. Her weight was 6400 g, around 4 SD below the mean for her age, and supine length was 63.5 cm, around 4 SD She was fed for a few months via a gastrostomy tube. An X-ray survey for skeletal anomalies was normal at the age of one year and bone age was normal. Cranial MRI at the age of three years, showed mild ventriculomegaly (Fig. 5) and magnetic resonance spectroscopy (MRS) measurings of NAA/ creatine, choline/creatine and lactate/creatine ratios were normal. An echocardiogram was normal. At four years of age, she was evaluated in our clinic for the first time. The child had friendly disposition. Her height was 82 cm (7 SD), weight was 10.8 kg (4.5 SD) and head circumference was 47 cm (2.5 SD). The metopic ridge was prominent. She had a fair complexion. Her hair was thin and scarce, the skin was rough, dry and warm without hyperpigmented lesions. No asymmetry of limbs or trunk was noticed. Dysmorphic features were noticed including broad forehead, midface retrusion, epicanthal folds, laterally sparse eyebrows, short nose, long philtrum, widely spaced teeth, micrognathia, long fingers, clinodactyly, and bilateral partial cutaneous syndactyly of the T2-3 (Figs. 1 and 2). She had no photosensitivity. Hepatomegaly or splenomegaly was not detected. Blood cholesterol level was 74 mg/dl. Developmental assessment: she smiled at 6 weeks, laughed at 3 months, reached out for toys at 4 months, rolled over at 6e8
289
Fig. 2. Patient 1. T2-3 syndactyly in both feet.
months, sat at 10 months. At the age of 4 years (when first evaluated by us), she walked with support of a walker and used ankle foot orthoses. Comprehension was limited to simple commands, expressive language was severely delayed. She had only twenty utterances and communicated mostly by gestures. She was hypotonic with normal reflexes. At the age of 9.5 years she had been assessed by a psychologist and was diagnosed with moderate intellectual disability (IQ-48). At the age of 10 years, her height was 107 cm (6 SD), weight was 17.7 kg (4.5 SD), and head circumference was 48.5 cm (3 SD). There was no improvement in her language, but her motor skills had improved and she could walk without support. Her facial features were coarser, with midface retrusion, small nose and widely spaced abnormally shaped secondary teeth (Figs. 3 and 4). No hepatosplenomegaly was detected. Ophthalmologic examination showed normal retinae and best visual acuity attained with correction was 10 (OD) and 10.25 (OS). No vitreous or corneal abnormalities were noted. Hearing was normal. SmitheLemlieOpitz syndrome was ruled out both by measurements of blood 7-dehydrocholesterol levels and sequencing of the DHCR7 gene. Repeated measurements of blood cholesterol levels at the age of 10 and 11 years were normal. 2.2. Patient 2
Fig. 1. Patient 1 at age 8 years. Note fair complexion, high, broad forehead, prominent metopic ridge, epicantal folds, depressed nasal bridge, small upturned nose, micrognathia.
This is the younger sister of patient 1. She was born by caesarean section at 35 weeks gestation. Her weight was 2400 g (w40th centile). Her supine length was 46.5 cm (3rde10th centile). There was premature rupture of membranes and delivery was by emergency caesarean section due to lack of progress in labor. Transient generalized edema was noticed immediately after birth, as were dysmorphic features similar to those of her older sister. She was diagnosed with hypothyroidism shortly after birth and was treated with L-thyroxin for a short while. She gained weight normally until the age of six months (she weighed 5580 g slightly below the 3rd centile); thereafter a marked deceleration of both weight and height were noticed but she did not need feeding gastrostomy like her sister. She was first evaluated at our clinic at the age of two years and five months. On examination, her supine length was 77 cm (mean height at this age is 91 cm and a standing height of 77 cm would be around 4 SD), weight was 8.45 kg (4 SD) and head circumference was 44.5 cm (3 SD). She was hypotonic and had similar dysmorphic features as her sister (Fig. 4). She had a fair complexion, like her sister and no photosensitivity. The skin was dry, rough, and warm without hyperpigmented lesions.
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Fig. 5. Midsagittal T1 weighted image of patient 1 shows thinning of the corpus callosum and mildly enlarged ventricle.
Fig. 3. Patient 1, age 11y, Note abnormal teeth.
No asymmetry of limbs or trunk was noticed. She had hepatosplenomegaly. The parents claimed that her normal body temperature was usually close to 38 C, but this finding was not rechecked by us. She had high myopia and strabismus. Since her blood cholesterol level was 55 mg/dl, she was treated with a high cholesterol diet with no improvement in growth or development. However cholesterol levels were normal around the age of two years even without the high cholesterol diet. An X-ray survey for skeletal anomalies was normal. Bone age was normal. She was seen again at the age of 7.5 years. At that time her supine length was 95 cm (mean height at this age is 124 cm and a
Fig. 4. The two sisters at the ages of 9 years (patient 1 Rt) and 7 years (patient 2 Lt). Note similar facial features.
standing height of 95 cm would be around 4.5 SD), weight was 13.6 kg (5 SD), and head circumference was 47.5 cm (3 SD). She did not attain any expressive language and was unable to understand even simple commands. She was unable to sit or walk. Her skin was normal, no hepatosplenomegaly was detected, and her parents reported normal body temperature. Eye examination at the age of nine years showed normal retinae and best visual acuity gained with a correction was 10.5 (OD) and 11 (OS). Hearing was normal. Blood cholesterol levels were normal (without treatment). Cranial MRI imaging showed ventriculomegaly and hypoplasia of the cerebellar vermis (Fig. 6). At the age of 7 years she had been assessed by a psychologist and was diagnosed with moderate to severe intellectual disability (IQ-38).
Fig. 6. Midsagittal T2 weighted image of patient 2 demonstrates thinning of the corpus callosum, enlarged third ventricle and atrophy of the vermis.
C. Vinkler et al. / European Journal of Medical Genetics 57 (2014) 288e292
3. Biochemical studies A thorough metabolic evaluation in patient 1 was done, including, blood glucose, carnitine, ammonia, lactate, pyruvate, amino acids, and very long chain fatty acids which were normal. Urinary, organic, mevalonic and amino acids were normal. Congenital viral infections tests were normal. A sweat test and serum immunoglobulins were normal. Negative serum transglutaminase antibodies were found which does not support the diagnosis of Celiac disease. Isoelectric focusing of transferrin was normal. Blood cholesterol levels were consistently low 70e80 mg/ dl (normal values 80e200 mg/dl). Both parents had normal plasma cholesterol and ApoB levels. Analyses of plasma bile acids by gas chromatography and mass spectrometry (GCeMS) were normal in both sisters, with no evidence of an inborn error of bile acid metabolism or a peroxisomal disorder. Plasma sterol analysis by GCeMS and analysis of urinary cholanoids by electrospray ionization tandem mass spectrometry (ESI-MS/MS) were normal (Table II). Quantification of sterols was performed in skin fibroblasts cultivated on a lipid depleted medium for 10 days to maximally stimulate cholesterol biosynthesis (courtesy of Dorothea Haas, Heidelberg). Sterols were then quantified by GCeMS (Table II). In patient 1 SmitheLemlieOpitz syndrome, desmosterolosis and ConradieHunermann syndrome were ruled out. In patient 2 the results were consistent with a low concentration of cholesterol and normal levels of most cholesterol precursors. The ratio of 7-DHC/Cholesterol was slightly elevated but not in the range of SmitheLemlieOpitz syndrome. The 8(9)-cholestanol and 8-DHC levels were quite prominent. The elevation of these metabolites could point to ConradieHunermann syndrome; however, the clinical symptoms were not typical of CDPX2, and 8(9)-cholestanol was completely normal in the sister who presented with similar clinical symptoms. Studies performed on a mitochondrial fraction from muscle biopsy, showed a partial complex 1 activity (30e40% of normal activity). CoQ levels were in the normal range. 4. Molecular studies The common Ashkenazi mutations of Bloom syndrome or familial dysautonomia were not found. Karyotype (resolution) was normal and no spontaneous chromosomal breakages were observed on chromosomal analysis. Molecular test for uniparental disomy of chromosome 7 was normal. Homozygosity mapping: DNA samples from the two affected and four healthy children and their parents were analyzed by Affymetrix Human Mapping 250k SNP Array. We found a few regions of shared homozygosity; the largest regions were ch. 3: 6.9 Mb (46,654,202e53,638,875), 4.5 Mb (128,740,840e 133,325,950) and ch. 6: 2.1 Mb (31,374,501e33,516,520). These regions contained more then 80 unknown genes. Since there was a partial complex 1 deficiency in the muscle biopsy from patient 1, and its assembly factor ACAD9 was found to be in the second homozygous region, we analyzed the gene and no sequence changes detected. 5. Discussion The clinical manifestations of these two patients included very short stature, microcephaly, severe intellectual disability, high myopia, and dysmorphic features with transient edema, hyperthermia, hepatosplenomegaly, and transient low cholesterol levels (Table 1). Neither girl developed any significant speech. No regression was observed and no crises were reported over time.
291
Table 1 Clinical, imaging findings in the study patients compared to SmitheLemlieOpitz syndrome. Feature
Patient 1 (at 4y)
Patient 2 (at 2y)
Smithe LemlieOpitz syndrome [Porter, 2008]
Short Stature Microcephaly Failure to gain weight Low birth weight Skin abnormalities Transient non-pitting edema Transient elevated body temperature High myopia Midface retrusion Epicanthal folds Anteverted nostrils Bitemporal narrowing Micrognathia Hypotonia Long fingers Clinodactyly Syndactyly T2-3 Intellectual disability (moderate esevere) Poor expressive language Hypotonia Strabismus Ptosis Vermis hypoplasia Ventriculomegaly Hypocholesterolemia (Normal values 80e200 mg) Congenital heart defect Hepatosplenomegaly Abnormal genitourinary system
þ (8 SD) þ (3 SD) þ þ þ þ
þ (7 SD) þ (3 SD) þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þþ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ74 mg/dl transient
þ þ þ þ þ þ55 mg/dl transient þ transient
þ þ þ þ þ þ þ þ
Dysmorphic features included midface retrusion, high, broad forehead, prominent metopic ridge, laterally sparse eyebrows, short nose, long philtrum, widely spaced abnormal teeth, long fingers, and partial cutaneous syndactyly of T2-3. Both had high myopia and strabismus. They had a fair complexion, which is marked in comparison with their healthy sibs. Both patients are females, there are no other affected individuals in the extended family, and no early miscarriages. The parents are both of Ashkenazi-Jewish origin and there is no vertical transmission. Hence, it is most likely that the condition is an autosomal recessive trait. The diagnosis of SmitheLemlieOpitz syndrome (OMIM 270400) was initially considered because of the combination of their dysmorphic features, low cholesterol levels, failure to thrive, and intellectual disability (Table 1). However, an extensive evaluation of cholesterol metabolism including measurement of 7dehydrocholesterol level did not show any abnormalities. In addition, sequence analysis of DHCR7 did not detect a mutation. Other disorders of cholesterol metabolism [Herman, 2003] were negated based on the clinical presentation of the sisters and normal metabolic studies (Table II). A possible defect in the transmembrane transport of cholesterol was ruled out by normal cholesterol transport studies in skin fibroblasts. Other possibilities were considered in the differential diagnosis. Dubowitz syndrome (OMIM 223370) was considered since it is characterized by short stature, microcephaly, dry skin and low blood cholesterol levels [Ahmad et al., 1999; Dubowitz, 1965]. However the patients reported here lacked the distinctive dysmorphic facial features characteristic of Dubowitz syndrome. Furthermore, linear growth and intellectual disability are usually not as severely affected in patients diagnosed with this
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syndrome, as was seen in these siblings. Dentici et al., described two siblings one of which had transient low cholesterol levels. Both had growth retardation and intellectual disability [Dentici et al., 2011]. However they had distinct facial dysmorphic features including blepharophimosis that our patient do not have. Syndromes associated with defects in DNA repair were considered because they are often associated with marked linear growth retardation, microcephaly, skin involvement, and intellectual disability [McKinnon and Caldecott, 2007]. However, these syndromes are typically associated with abnormalities of the immune system, radiation hypersensitivity, and occasionally a predisposition to cancer [Cleaver et al., 2009] that our patients did not have. Furthermore, no spontaneous chromosomal breakages were observed on chromosomal analysis. Both patients had low blood cholesterol levels during their first years of life. Defects in cholesterol metabolism as in SmitheLemlie Opitz syndrome [Smith et al., 1964] and others, affect linear growth via interruption of early developmental pathways [Herman, 2003]. It is known that cholesterol deficiencies during embryogenesis and organogenesis cause severe abnormalities [Roux et al., 2000]. Cholesterol is a key constituent of the cell membrane, the structure of which is important in cell-to-cell interactions, which are essential in embryonic differentiation. Cholesterol not only plays a role in the physical structure of the membrane (e.g., its viscosity and interference with phospholipids), but also constitutes specific parts of this membrane, such as the caveolae (detergentresistant domains), which are important for transduction and ceramide-induced apoptosis [Brown and London, 1998]. The sonic hedgehog (SHH) pathway is critical in embryogenesis. The SHH protein is a morphogen central to growth, patterning, and morphogenesis [Ingham and McMahon, 2001]. The SHH protein needs to be modified by cholesterol in order to be active. Lack of cholesterol leads to defective SHH signaling, causing abnormal brain development and affects other organs as well [Lanoue et al., 1997; Repetto et al., 1990]. Previous studies on the teratogenic effect of a low cholesterol environment show that lack of cholesterol rather than accumulation of aberrant sterols causes various congenital malformations in mice embryos [Gaoua et al., 2000]. Even though defects in SHH pathway are known to be associated with limb and other congenital anomalies missing in the patients reported here, it is tempting to postulate that embryogenesis occurred in a cholesterol-poor milieu, that resulted in the typical dysmorphic features and abnormal brain development (vermian hypoplasia). It is difficult to explain why abnormal linear growth only started postnatally in both patients. The transient nature of low cholesterol levels, as well as the nonprogressive course of this syndrome may indicate that low cholesterol levels were secondary to a defective pathway that could be the underlying cause of this syndrome. Another possible cause
might be a faulty transition of a prenatal cholesterol metabolic pathway to a mature pathway. Homozygosity mapping did not identify an obvious candidate gene. Exome sequencing may enable the discovery of the underlying causative gene, which we propose to be the cause of this newly recognized syndrome inherited in an autosomal recessive pattern. Acknowledgment We thank Professor Peter Clayton for performing the biochemical analyses. We thank the patients’ family for kindly allowing us to publish this report and for their cooperation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmg.2014.03.010. References Ahmad A, Amalfitano A, Chen YT, Kishnani PS, Miller C, Kelley R. Dubowitz syndrome: a defect in the cholesterol biosynthetic pathway? Am J Med Genet 1999;86:503e4. Brown DA, London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 1998;14:111e36. Cleaver JE, Lam ET, Revet I. Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity. Nat Rev Genet 2009;10:756e68. Dentici ML, Mingarelli R, Dallapiccola B. The difficult nosology of blepharophimosismental retardation syndromes: report on two siblings. Am J Med Genet A 2011;155A:459e65. Dubowitz V. Familial low birth weight dwarfism with an unusual facies and a skin eruption. J Med Genet 1965;2:12e7. Gaoua W, Wolf C, Chevy F, Ilien F, Roux C. Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smithe LemlieOpitz syndrome animal model. J Lipid Res 2000;41:637e46. Hall JG. Review and hypothesis: syndromes with severe intrauterine growth restriction and very short stature are they related to the epigenetic mechanism(s) of fetal survival involved in the developmental origins of adult health and disease? Am J Med Genet A 2010;152A:512e7. Herman GE. Disorders of cholesterol biosynthesis: prototypic metabolic malformation syndromes. Hum Mol Genet 2003;12:75e88. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 2001;15:3059e87. Lanoue L, Dehart DB, Hinsdale ME, Maeda N, Tint GS, Sulik KK. Limb, genital, CNS, and facial malformations result from gene/environment-induced cholesterol deficiency: further evidence for a link to sonic hedgehog. Am J Med Genet 1997;73:24e31. McKinnon PJ, Caldecott KW. DNA strand break repair and human genetic disease. Ann Rev Genomics Hum Genet 2007;8:37e55. Porter FD. SmitheLemlieOpitz syndrome: pathogenesis, diagnosis and management. Eur J Hum Genet 2008;16:535e41. Roux C, Wolf C, Mulliez N, Gaoua W, Cormier V, Chevy F, et al. Role of cholesterol in embryonic development. Am J Clin Nutr 2000;71(5Suppl.):1270Se9S. Repetto M, Maziere JC, Citadelle D, Dupuis R, Meier M, Biade S, et al. Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro. Teratology 1990;42:611e8. Smith DW, Lemli D, Opitz JM. A newly recognized syndrome of multiple congenital anomalies. J Pediatr 1964;64:210e7.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
New clinically defined syndrome
A newly recognized syndrome of severe growth deficiency, microcephaly, intellectual disability, and characteristic facial features Chana Vinkler a, b, *, Esther Leshinsky-Silver b, c, e, Marina Michelson a, b, Dorothea Haas f, Tally Lerman-Sagie b, d, e, Dorit Lev a, b, e a
Institute of Medical Genetics, Wolfson Medical Center, Holon, Israel Metabolic Neurogenetic Service, Wolfson Medical Center, Holon, Israel Molecular Genetics Laboratory, Wolfson Medical Center, Holon, Israel d Pediatric Neurology Unit, Wolfson Medical Center, Holon, Israel e Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel f Division of Inborn Metabolic Diseases, University Children’s Hospital, Im Neuenheimer Feld, Heidelberg, Germany b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2013 Accepted 25 March 2014 Available online 5 April 2014
Genetic syndromes with proportionate severe short stature are rare. We describe two sisters born to nonconsanguineous parents with severe linear growth retardation, poor weight gain, microcephaly, characteristic facial features, cutaneous syndactyly of the toes, high myopia, and severe intellectual disability. During infancy and early childhood, the girls had transient hepatosplenomegaly and low blood cholesterol levels that normalized later. A thorough evaluation including metabolic studies, radiological, and genetic investigations were all normal. Cholesterol metabolism and transport were studied and no definitive abnormality was found. No clinical deterioration was observed and no metabolic crises were reported. After due consideration of other known hereditary causes of post-natal severe linear growth retardation, microcephaly, and intellectual disability, we propose that this condition represents a newly recognized autosomal recessive multiple congenital anomaly-intellectual disability syndrome. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Microcephaly Small stature Intellectual disability Low cholesterol
1. Introduction
2. Patients report
Genetic syndromes involving severe postnatal proportionate short stature are rare. Numerous etiologies should be considered when investigating patients who present with familial severe growth retardation and systemic involvement. As was previously discussed [Hall, 2010], these include metabolic diseases, congenital anomalies, endocrine disorders, nutritional factors, exposure to teratogens, exposure to congenital infections, chromosomal aberrations, and many genetic disorders. We present two sisters with postnatal proportionate short stature, microcephaly, high myopia, severe intellectual disability, dysmorphic features, and transient low cholesterol levels. We suggest that this is a newly recognized syndrome probably inherited in an autosomal recessive pattern.
2.1. Patient 1
* Corresponding author. Institute of Medical Genetics, Wolfson Medical Center, Holon 58100, Israel. Tel.: þ97 235028536; fax: þ97 235028566. E-mail addresses: [email protected] (C. Vinkler), [email protected] (D. Lev). http://dx.doi.org/10.1016/j.ejmg.2014.03.010 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
The patient was the first child of seven children. Five of the children (three boys and two girls) have normal growth and development. She was born to a healthy 22-year-old woman and her unrelated 23-year-old husband, both of Ashkenazi Jewish ancestry. They had no early miscarriages. The pregnancy was uneventful and she was born at term via an uncomplicated delivery. Her birth weight was 2930 g (w15th centile), her supine length was 47 cm (w5th centile) and her head circumference was 33 cm (w10th centile). Dysmorphic features were noticed at birth including short nose, depressed nasal bridge, low set ears, short neck, clinodactyly, and cutaneous syndactyly of T2-3. Mild edema of the upper thorax, hypotonia, and strabismus were also noted. Cranial ultrasound showed an intraventricular hemorrhage (grade IIeIII) and caudothalamic cysts. Thyroid function tests were normal. Two weeks later, a repeat cranial ultrasound was normal and no cysts were demonstrated. Around the age of 6 months growth retardation had been noted. Her weight was 5150 g, around 3 SD
C. Vinkler et al. / European Journal of Medical Genetics 57 (2014) 288e292
below the mean for her age, and supine length was 58 cm, around 4 SD below the mean for her age. At the age of one year, she was hospitalized due to severe failure to thrive. Her weight was 6400 g, around 4 SD below the mean for her age, and supine length was 63.5 cm, around 4 SD She was fed for a few months via a gastrostomy tube. An X-ray survey for skeletal anomalies was normal at the age of one year and bone age was normal. Cranial MRI at the age of three years, showed mild ventriculomegaly (Fig. 5) and magnetic resonance spectroscopy (MRS) measurings of NAA/ creatine, choline/creatine and lactate/creatine ratios were normal. An echocardiogram was normal. At four years of age, she was evaluated in our clinic for the first time. The child had friendly disposition. Her height was 82 cm (7 SD), weight was 10.8 kg (4.5 SD) and head circumference was 47 cm (2.5 SD). The metopic ridge was prominent. She had a fair complexion. Her hair was thin and scarce, the skin was rough, dry and warm without hyperpigmented lesions. No asymmetry of limbs or trunk was noticed. Dysmorphic features were noticed including broad forehead, midface retrusion, epicanthal folds, laterally sparse eyebrows, short nose, long philtrum, widely spaced teeth, micrognathia, long fingers, clinodactyly, and bilateral partial cutaneous syndactyly of the T2-3 (Figs. 1 and 2). She had no photosensitivity. Hepatomegaly or splenomegaly was not detected. Blood cholesterol level was 74 mg/dl. Developmental assessment: she smiled at 6 weeks, laughed at 3 months, reached out for toys at 4 months, rolled over at 6e8
289
Fig. 2. Patient 1. T2-3 syndactyly in both feet.
months, sat at 10 months. At the age of 4 years (when first evaluated by us), she walked with support of a walker and used ankle foot orthoses. Comprehension was limited to simple commands, expressive language was severely delayed. She had only twenty utterances and communicated mostly by gestures. She was hypotonic with normal reflexes. At the age of 9.5 years she had been assessed by a psychologist and was diagnosed with moderate intellectual disability (IQ-48). At the age of 10 years, her height was 107 cm (6 SD), weight was 17.7 kg (4.5 SD), and head circumference was 48.5 cm (3 SD). There was no improvement in her language, but her motor skills had improved and she could walk without support. Her facial features were coarser, with midface retrusion, small nose and widely spaced abnormally shaped secondary teeth (Figs. 3 and 4). No hepatosplenomegaly was detected. Ophthalmologic examination showed normal retinae and best visual acuity attained with correction was 10 (OD) and 10.25 (OS). No vitreous or corneal abnormalities were noted. Hearing was normal. SmitheLemlieOpitz syndrome was ruled out both by measurements of blood 7-dehydrocholesterol levels and sequencing of the DHCR7 gene. Repeated measurements of blood cholesterol levels at the age of 10 and 11 years were normal. 2.2. Patient 2
Fig. 1. Patient 1 at age 8 years. Note fair complexion, high, broad forehead, prominent metopic ridge, epicantal folds, depressed nasal bridge, small upturned nose, micrognathia.
This is the younger sister of patient 1. She was born by caesarean section at 35 weeks gestation. Her weight was 2400 g (w40th centile). Her supine length was 46.5 cm (3rde10th centile). There was premature rupture of membranes and delivery was by emergency caesarean section due to lack of progress in labor. Transient generalized edema was noticed immediately after birth, as were dysmorphic features similar to those of her older sister. She was diagnosed with hypothyroidism shortly after birth and was treated with L-thyroxin for a short while. She gained weight normally until the age of six months (she weighed 5580 g slightly below the 3rd centile); thereafter a marked deceleration of both weight and height were noticed but she did not need feeding gastrostomy like her sister. She was first evaluated at our clinic at the age of two years and five months. On examination, her supine length was 77 cm (mean height at this age is 91 cm and a standing height of 77 cm would be around 4 SD), weight was 8.45 kg (4 SD) and head circumference was 44.5 cm (3 SD). She was hypotonic and had similar dysmorphic features as her sister (Fig. 4). She had a fair complexion, like her sister and no photosensitivity. The skin was dry, rough, and warm without hyperpigmented lesions.
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C. Vinkler et al. / European Journal of Medical Genetics 57 (2014) 288e292
Fig. 5. Midsagittal T1 weighted image of patient 1 shows thinning of the corpus callosum and mildly enlarged ventricle.
Fig. 3. Patient 1, age 11y, Note abnormal teeth.
No asymmetry of limbs or trunk was noticed. She had hepatosplenomegaly. The parents claimed that her normal body temperature was usually close to 38 C, but this finding was not rechecked by us. She had high myopia and strabismus. Since her blood cholesterol level was 55 mg/dl, she was treated with a high cholesterol diet with no improvement in growth or development. However cholesterol levels were normal around the age of two years even without the high cholesterol diet. An X-ray survey for skeletal anomalies was normal. Bone age was normal. She was seen again at the age of 7.5 years. At that time her supine length was 95 cm (mean height at this age is 124 cm and a
Fig. 4. The two sisters at the ages of 9 years (patient 1 Rt) and 7 years (patient 2 Lt). Note similar facial features.
standing height of 95 cm would be around 4.5 SD), weight was 13.6 kg (5 SD), and head circumference was 47.5 cm (3 SD). She did not attain any expressive language and was unable to understand even simple commands. She was unable to sit or walk. Her skin was normal, no hepatosplenomegaly was detected, and her parents reported normal body temperature. Eye examination at the age of nine years showed normal retinae and best visual acuity gained with a correction was 10.5 (OD) and 11 (OS). Hearing was normal. Blood cholesterol levels were normal (without treatment). Cranial MRI imaging showed ventriculomegaly and hypoplasia of the cerebellar vermis (Fig. 6). At the age of 7 years she had been assessed by a psychologist and was diagnosed with moderate to severe intellectual disability (IQ-38).
Fig. 6. Midsagittal T2 weighted image of patient 2 demonstrates thinning of the corpus callosum, enlarged third ventricle and atrophy of the vermis.
C. Vinkler et al. / European Journal of Medical Genetics 57 (2014) 288e292
3. Biochemical studies A thorough metabolic evaluation in patient 1 was done, including, blood glucose, carnitine, ammonia, lactate, pyruvate, amino acids, and very long chain fatty acids which were normal. Urinary, organic, mevalonic and amino acids were normal. Congenital viral infections tests were normal. A sweat test and serum immunoglobulins were normal. Negative serum transglutaminase antibodies were found which does not support the diagnosis of Celiac disease. Isoelectric focusing of transferrin was normal. Blood cholesterol levels were consistently low 70e80 mg/ dl (normal values 80e200 mg/dl). Both parents had normal plasma cholesterol and ApoB levels. Analyses of plasma bile acids by gas chromatography and mass spectrometry (GCeMS) were normal in both sisters, with no evidence of an inborn error of bile acid metabolism or a peroxisomal disorder. Plasma sterol analysis by GCeMS and analysis of urinary cholanoids by electrospray ionization tandem mass spectrometry (ESI-MS/MS) were normal (Table II). Quantification of sterols was performed in skin fibroblasts cultivated on a lipid depleted medium for 10 days to maximally stimulate cholesterol biosynthesis (courtesy of Dorothea Haas, Heidelberg). Sterols were then quantified by GCeMS (Table II). In patient 1 SmitheLemlieOpitz syndrome, desmosterolosis and ConradieHunermann syndrome were ruled out. In patient 2 the results were consistent with a low concentration of cholesterol and normal levels of most cholesterol precursors. The ratio of 7-DHC/Cholesterol was slightly elevated but not in the range of SmitheLemlieOpitz syndrome. The 8(9)-cholestanol and 8-DHC levels were quite prominent. The elevation of these metabolites could point to ConradieHunermann syndrome; however, the clinical symptoms were not typical of CDPX2, and 8(9)-cholestanol was completely normal in the sister who presented with similar clinical symptoms. Studies performed on a mitochondrial fraction from muscle biopsy, showed a partial complex 1 activity (30e40% of normal activity). CoQ levels were in the normal range. 4. Molecular studies The common Ashkenazi mutations of Bloom syndrome or familial dysautonomia were not found. Karyotype (resolution) was normal and no spontaneous chromosomal breakages were observed on chromosomal analysis. Molecular test for uniparental disomy of chromosome 7 was normal. Homozygosity mapping: DNA samples from the two affected and four healthy children and their parents were analyzed by Affymetrix Human Mapping 250k SNP Array. We found a few regions of shared homozygosity; the largest regions were ch. 3: 6.9 Mb (46,654,202e53,638,875), 4.5 Mb (128,740,840e 133,325,950) and ch. 6: 2.1 Mb (31,374,501e33,516,520). These regions contained more then 80 unknown genes. Since there was a partial complex 1 deficiency in the muscle biopsy from patient 1, and its assembly factor ACAD9 was found to be in the second homozygous region, we analyzed the gene and no sequence changes detected. 5. Discussion The clinical manifestations of these two patients included very short stature, microcephaly, severe intellectual disability, high myopia, and dysmorphic features with transient edema, hyperthermia, hepatosplenomegaly, and transient low cholesterol levels (Table 1). Neither girl developed any significant speech. No regression was observed and no crises were reported over time.
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Table 1 Clinical, imaging findings in the study patients compared to SmitheLemlieOpitz syndrome. Feature
Patient 1 (at 4y)
Patient 2 (at 2y)
Smithe LemlieOpitz syndrome [Porter, 2008]
Short Stature Microcephaly Failure to gain weight Low birth weight Skin abnormalities Transient non-pitting edema Transient elevated body temperature High myopia Midface retrusion Epicanthal folds Anteverted nostrils Bitemporal narrowing Micrognathia Hypotonia Long fingers Clinodactyly Syndactyly T2-3 Intellectual disability (moderate esevere) Poor expressive language Hypotonia Strabismus Ptosis Vermis hypoplasia Ventriculomegaly Hypocholesterolemia (Normal values 80e200 mg) Congenital heart defect Hepatosplenomegaly Abnormal genitourinary system
þ (8 SD) þ (3 SD) þ þ þ þ
þ (7 SD) þ (3 SD) þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þþ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ74 mg/dl transient
þ þ þ þ þ þ55 mg/dl transient þ transient
þ þ þ þ þ þ þ þ
Dysmorphic features included midface retrusion, high, broad forehead, prominent metopic ridge, laterally sparse eyebrows, short nose, long philtrum, widely spaced abnormal teeth, long fingers, and partial cutaneous syndactyly of T2-3. Both had high myopia and strabismus. They had a fair complexion, which is marked in comparison with their healthy sibs. Both patients are females, there are no other affected individuals in the extended family, and no early miscarriages. The parents are both of Ashkenazi-Jewish origin and there is no vertical transmission. Hence, it is most likely that the condition is an autosomal recessive trait. The diagnosis of SmitheLemlieOpitz syndrome (OMIM 270400) was initially considered because of the combination of their dysmorphic features, low cholesterol levels, failure to thrive, and intellectual disability (Table 1). However, an extensive evaluation of cholesterol metabolism including measurement of 7dehydrocholesterol level did not show any abnormalities. In addition, sequence analysis of DHCR7 did not detect a mutation. Other disorders of cholesterol metabolism [Herman, 2003] were negated based on the clinical presentation of the sisters and normal metabolic studies (Table II). A possible defect in the transmembrane transport of cholesterol was ruled out by normal cholesterol transport studies in skin fibroblasts. Other possibilities were considered in the differential diagnosis. Dubowitz syndrome (OMIM 223370) was considered since it is characterized by short stature, microcephaly, dry skin and low blood cholesterol levels [Ahmad et al., 1999; Dubowitz, 1965]. However the patients reported here lacked the distinctive dysmorphic facial features characteristic of Dubowitz syndrome. Furthermore, linear growth and intellectual disability are usually not as severely affected in patients diagnosed with this
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syndrome, as was seen in these siblings. Dentici et al., described two siblings one of which had transient low cholesterol levels. Both had growth retardation and intellectual disability [Dentici et al., 2011]. However they had distinct facial dysmorphic features including blepharophimosis that our patient do not have. Syndromes associated with defects in DNA repair were considered because they are often associated with marked linear growth retardation, microcephaly, skin involvement, and intellectual disability [McKinnon and Caldecott, 2007]. However, these syndromes are typically associated with abnormalities of the immune system, radiation hypersensitivity, and occasionally a predisposition to cancer [Cleaver et al., 2009] that our patients did not have. Furthermore, no spontaneous chromosomal breakages were observed on chromosomal analysis. Both patients had low blood cholesterol levels during their first years of life. Defects in cholesterol metabolism as in SmitheLemlie Opitz syndrome [Smith et al., 1964] and others, affect linear growth via interruption of early developmental pathways [Herman, 2003]. It is known that cholesterol deficiencies during embryogenesis and organogenesis cause severe abnormalities [Roux et al., 2000]. Cholesterol is a key constituent of the cell membrane, the structure of which is important in cell-to-cell interactions, which are essential in embryonic differentiation. Cholesterol not only plays a role in the physical structure of the membrane (e.g., its viscosity and interference with phospholipids), but also constitutes specific parts of this membrane, such as the caveolae (detergentresistant domains), which are important for transduction and ceramide-induced apoptosis [Brown and London, 1998]. The sonic hedgehog (SHH) pathway is critical in embryogenesis. The SHH protein is a morphogen central to growth, patterning, and morphogenesis [Ingham and McMahon, 2001]. The SHH protein needs to be modified by cholesterol in order to be active. Lack of cholesterol leads to defective SHH signaling, causing abnormal brain development and affects other organs as well [Lanoue et al., 1997; Repetto et al., 1990]. Previous studies on the teratogenic effect of a low cholesterol environment show that lack of cholesterol rather than accumulation of aberrant sterols causes various congenital malformations in mice embryos [Gaoua et al., 2000]. Even though defects in SHH pathway are known to be associated with limb and other congenital anomalies missing in the patients reported here, it is tempting to postulate that embryogenesis occurred in a cholesterol-poor milieu, that resulted in the typical dysmorphic features and abnormal brain development (vermian hypoplasia). It is difficult to explain why abnormal linear growth only started postnatally in both patients. The transient nature of low cholesterol levels, as well as the nonprogressive course of this syndrome may indicate that low cholesterol levels were secondary to a defective pathway that could be the underlying cause of this syndrome. Another possible cause
might be a faulty transition of a prenatal cholesterol metabolic pathway to a mature pathway. Homozygosity mapping did not identify an obvious candidate gene. Exome sequencing may enable the discovery of the underlying causative gene, which we propose to be the cause of this newly recognized syndrome inherited in an autosomal recessive pattern. Acknowledgment We thank Professor Peter Clayton for performing the biochemical analyses. We thank the patients’ family for kindly allowing us to publish this report and for their cooperation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmg.2014.03.010. References Ahmad A, Amalfitano A, Chen YT, Kishnani PS, Miller C, Kelley R. Dubowitz syndrome: a defect in the cholesterol biosynthetic pathway? Am J Med Genet 1999;86:503e4. Brown DA, London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 1998;14:111e36. Cleaver JE, Lam ET, Revet I. Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity. Nat Rev Genet 2009;10:756e68. Dentici ML, Mingarelli R, Dallapiccola B. The difficult nosology of blepharophimosismental retardation syndromes: report on two siblings. Am J Med Genet A 2011;155A:459e65. Dubowitz V. Familial low birth weight dwarfism with an unusual facies and a skin eruption. J Med Genet 1965;2:12e7. Gaoua W, Wolf C, Chevy F, Ilien F, Roux C. Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smithe LemlieOpitz syndrome animal model. J Lipid Res 2000;41:637e46. Hall JG. Review and hypothesis: syndromes with severe intrauterine growth restriction and very short stature are they related to the epigenetic mechanism(s) of fetal survival involved in the developmental origins of adult health and disease? Am J Med Genet A 2010;152A:512e7. Herman GE. Disorders of cholesterol biosynthesis: prototypic metabolic malformation syndromes. Hum Mol Genet 2003;12:75e88. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 2001;15:3059e87. Lanoue L, Dehart DB, Hinsdale ME, Maeda N, Tint GS, Sulik KK. Limb, genital, CNS, and facial malformations result from gene/environment-induced cholesterol deficiency: further evidence for a link to sonic hedgehog. Am J Med Genet 1997;73:24e31. McKinnon PJ, Caldecott KW. DNA strand break repair and human genetic disease. Ann Rev Genomics Hum Genet 2007;8:37e55. Porter FD. SmitheLemlieOpitz syndrome: pathogenesis, diagnosis and management. Eur J Hum Genet 2008;16:535e41. Roux C, Wolf C, Mulliez N, Gaoua W, Cormier V, Chevy F, et al. Role of cholesterol in embryonic development. Am J Clin Nutr 2000;71(5Suppl.):1270Se9S. Repetto M, Maziere JC, Citadelle D, Dupuis R, Meier M, Biade S, et al. Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro. Teratology 1990;42:611e8. Smith DW, Lemli D, Opitz JM. A newly recognized syndrome of multiple congenital anomalies. J Pediatr 1964;64:210e7.
