BACKGROUND Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. NBS-1, the gene defective in NBS, is located on chromosome 8q21 and has recently been cloned. The gene product, nibrin, is a novel protein, which is member of the hMre11/hRad50 protein complex, suggesting that the gene is involved in DNA double strand break repair.
AIMS To study the clinical and laboratory features of NBS as well as the genotype–phenotype relation.
METHODS Fifty five patients with NBS, included in the NBS registry in Nijmegen were evaluated. The majority of the patients were of eastern European ancestry. Most of them had shown a truncating 5 bp deletion 657–661 delACAAA. Four further truncating mutations have been identified in patients with other distinct haplotypes.
RESULTS AND CONCLUSIONS Essential features found in NBS were microcephaly, usually without severe retardation, typical facial appearance, immunodeficiency, chromosomal instability, x ray hypersensitivity, and predisposition to malignancy. In 40% of the patients cancer was noted before the age of 21 years. Important additional features were skin abnormalities, particularly café au lait spots and vitiligo, and congenital malformations, particularly clinodactyly and syndactyly. Congenital malformations, immunodeficiency, radiation hypersensitivity, and cancer predispostion were comprehensible in case of dysfunctioning of DNA repair mechanisms. No specific genotype–phenotype relation could be found. Patients with the same genotype may show different phenotypes and patients with different genotypes may express the same phenotype. Specific mutations did not lead to specific clinical features.
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Nijmegen breakage syndrome (NBS) is a rare autosomal recessive condition, which belongs to the so called DNA repair disorders, which also include Bloom syndrome, xeroderma pigmentosa, Fanconi anaemia, and ataxia telangiectasia. These disorders show overlapping clinical and cell biological features, but are genetically heterogeneous.1-29 Spontaneous chromosomal instability, predisposition to cancer, and immunodeficiency are characteristic features of NBS, Bloom syndrome, and ataxia telangiectasia.1-29
The gene responsible for NBS, NBS-1, is located on chromosome 8q21 and has recently been identified.26 27 The encoding protein has been named nibrin. The domains found in nibrin and the NBS phenotype suggest that NBS is caused by defective responses to DNA double strand breaks.27 28
In this report we provide an extended follow up of the first recognised NBS patient described in 1981 and evaluate the clinical and laboratory features of 55 NBS patients included in our NBS registry in Nijmegen.
This boy (fig 1) is the sixth child of consanguineous healthy parents (second cousins). He was born in 1969 after a pregnancy of 38 weeks duration. Pregnancy was complicated by pyelonephritis at 24 weeks. Birth weight was 2500 g (P10) (data concerning length and head circumference at birth not available). The neonatal period was normal. When he was 6 weeks old his head circumference was 33 cm (<P3). Development of language skills was delayed. During infancy he suffered from recurrent upper respiratory tract infections, chickenpox, and measles. Special education was necessary because of learning difficulties and hyperactivity. We examined him aged 9, when we noted a small microcephalic boy with head circumference 45 cm (<P3), height 124 cm (<P3), and weight 21 kg (<P3). He had a typical face with a receding forehead, prominent midface with long nose, and a receding mandible. Sun sensitive erythema of the face and many freckles were noted as well as café au lait spots and patches of vitiligo. TIQ was 67. Neurological examination was otherwise unremarkable. EEG revealed no abnormalities.
Routine blood tests were normal, as were endocrinological studies. Immunological studies showed IgA and IgE deficiency with normal IgG and IgM (IgA <0.05 g/l, IgE <1 IU/ml, IgG 11.30 g/l, IgM 0.69 g/l); cellular immunity was normal. Cytogenetic studies revealed multiple rearrangements of chromosomes 7 and 14. We proposed a provisional name of Nijmegen breakage syndrome (NBS).1 At 13 years of age mild thoracolumbar scoliosis was noted. At 14 years of age he experienced repeated skin infections and suffered a severe bronchopneumonia. Head circumference remained below P3 (fig 2). At the age of 17, repeat immunological studies showed undetectable serum IgA and serum IgE concentrations, and disturbances in synthesis of specific antibodies as well as some disturbances in cellular immunity. At the age of 19 he was admitted with malaise and fever. Anx ray of the chest revealed a pathological mediastinal mass, which proved to be non-Hodgkin lymphoma. He received chemotherapy with prednisone and vincristine followed by maintenance with methotrexate. The latter had to be discontinued after a year because of vomiting and loss of weight. The disease remitted. At the age of 24, human immunoglobulin substitution was started subcutaneously because of agammaglobulinaemia (IgG 1.37 g/l, IgM 0.22 g/l). At 27 years of age he had repeated fits with lowered consciousness. Neurological examination was unremarkable. EEG showed low voltage activity, no epileptic discharges. A hyperventilation provocation test was strongly positive. Breathing control manoeuvres were beneficial. He is currently 30 and has no specific complaints.
Clinical and laboratory features of NBS
PATIENTS AND METHODS
We conducted a computer aided literature search to obtain data on patients with NBS syndrome.1-11 Supplementary information was obtained from questionnaires completed by the patients' attending physicians. We now report on the available clinical and laboratory features of the 55 patients included in the NBS registry to date.
The disease appears prevalent among those of eastern and central European origin, particularly among Polish people. The 55 patients comprise 31 men and 24 women. Of the 55 patients, 36 are still alive; the oldest is now 30 years old (table 1).
Growth and development
All patient are microcephalic. Head circumference at birth varied between 26.5 and 36 cm. About 75% of patients had a birth head circumference below the 3rd centile, so not all were microcephalic at birth. However, all developed progressive and severe microcephaly during the first months of life. They have early growth retardation, height falling below the 10th centile in all. The growth retardation is proportionate and weight corresponds to height. Feeding difficulties are often reported in infancy, probably as a result of abnormal development of the mandible (see below).
Developmental milestones were generally reached at normal times during the first year of life. Hyperactivity was common. Mental development is normal in 40%, 50% have borderline to mild retardation, while 10% are moderately retarded. Severe mental retardation has not been reported. We found no correlation between head circumference at birth and mental development.
All patients have a typical distinctive facial appearance, characterised by a receding forehead, prominent mid face with long nose and long philtrum, receding mandible, upward slanting palpebral fissures usually accompanied by epicanthic folds, freckles on the cheeks and nose, large ears with dysplastic helices, and sparse hair (fig 1). These characteristics become more obvious with age. Subtle scleral telangiectasia is seen in some.
Café au lait spots, vitiligo, sun sensitivity of the eyelids, and pigment deposits in the fundus of the eye are comon. Cutaneous telangiectasia is seen ocassionally.
The most common malformations are clinodactyly and/or syndactyly, noted in about 50% of the patients. Less common are anal atresia/stenosis (patients 9, 10, 31, 45), ovarian dysgenesis (patients 8, 9, 20, 26, 53), hydronephrosis (patients 2, 7, 11), and hip dysplasia (patients 11, 28, 49, 53). Other malformations reported are hypoplastic trachea (patient 50), cavernous angioma (patient 46), agenesis of phalanges (patient 34), hypospadias (patient 37), left renal hypoplasia (patient 45), and single kidney (patient 41). Cerebral malformations reported are schizencephaly (patient 37), occipital cyst (patient 17), and hydrocephalus (patients 11 and 46).
Infections are common, most frequently of the respiratory tract followed by urinary tract infections. Gastrointestinal infections are reported relatively infrequently. The infections are typically community acquired infections rather than opportunistic infections.
To date 22 patients, varying in age from 1 to 22 years have developed a malignancy. Of these, 16 developed a lymphoma (patients 1, 3, 4, 5, 9, 11, 13, 16, 20, 27, 28, 35, 38, 39, 42, 49), the majority B cell. The remaining six have leukaemia (patient 54), precursor T cell lymphoblastic lymphoma/leukaemia (TLBL/ALL) (patients 29 and 51), glioma (patient 7), medulloblastoma (patient 34), or rhabdomyosarcoma.37 Eight of the 22 patients with malignancy are still alive, including the patient described in this case report.
α Fetoprotein concentrations
In contrast to the situation with ataxia telangiectasia, all NBS patients had normal serum α fetoprotein concentrations.
Figure 3 shows concentrations of IgG, IgA, and IgM, measured in 48 patients. The most commonly reported defects in humoral immunity were IgG and IgA deficiency, as well as IgG2 and IgG4 deficiency. IgM deficiency was very rare.
Agammaglobulinaemia was found in 16 patients (pateints 2, 3, 5, 8, 17, 18, 27, 29, 31, 36, 40, 41, 43, 46, 48, 55). Selective IgA deficiency was seen in five patients (patients 1, 9, 10, 14, 54). IgA deficiency as well as IgG2 deficiency, with or without IgG4 deficiency was noted in seven patients (patients 13, 25, 28, 34, 38, 42, 47). IgG2 deficiency with or without IgG4 deficiency was seen in 11 patients (patients 7, 11, 19, 20, 21, 22, 23, 24, 35, 52, 53). Selective IgG4 deficiency was noted in three patients (patients 32, 45, 51). Only six patients had normal immunoglobulins (patients 12, 26, 30, 37, 49, 50). For some patients not all data were available: in four patients with IgA deficiency, IgG subclasses were not determined; in three patients with normal immunoglobulins, IgG subclasses were not determined. Figure4 shows the percentages of CD3, CD4, and CD8 cells of about 70% of the patients. The most commonly reported defects in cellular immunity were reduced percentages of total CD3+ cells and CD4+ cells and a decreased CD4+/CD8+ ratio. The frequency of CD8+ cells usually does not appear to be affected. The in vitro response of lymphocytes to mitogenic stimuli such as phytohaemagglutinin (PHA) was decreased in nearly all NBS patients tested (of 51 patients tested, 47 had a greatly reduced response, two patients had a slightly reduced response, and two had a normal response).
Constitutional karyotypes of NBS patients are normal. Cultured T cells often show a poor proliferative capacity, making cytogenetic analysis far from easy. In all patients cytogenetic aberrations are present in 10–45% of metaphases from PHA cultured T cells. Most of the rearrangements occur preferentially in chromosomes 7 and 14 and are typically inversions and translocations, with breakpoints at the sites of immunoglobulin or T cell receptor genes. Inv(7)(p13q35) is the most frequently detected aberration in NBS. Other frequent rearrangements are t(7;14)(p13;q11), t(7;14)(q35;q11), t(7;7)(p13;q35), and t(14;14)(q11;q32).5 7 11 15 16 Chromosomal rearrangements typically are not increased in cultured fibroblasts from NBS patients and, in cases where increased rearrangements in fibroblasts are reported, no bias in the sites of rearrangement analogous to that in lymphocytes is observed.
Radiation hypersensitivity and radioresistant DNA synthesis
Primary cells (fibroblasts and lymphocytes) cultured from NBS patients typically display poor growth in culture, a feature they share with cells from ataxia telangiectasia patients. In addition, primary cells and transformed cells have aberrant responses to ionising radiation. In colony forming assays, NBS cells are 3–5 times more sensitive to ionising radiation or radiomimetic drugs than normal cells. Radiation induced chromosome aberrations occur with increased frequency in NBS cells. x Ray hypersensitivity was present in 26 of 27 patients tested (patients 1, 3, 5, 7–12, 14, 18, 20–24, 27–29, 32, 33, 36, 37, 45, 47, 53). One patient showed intermediate sensitivity (patient 42). NBS cells also display radioresistant DNA synthesis (RDS), the inability to halt or slow S phase progression after exposure to high doses ofx rays. The inhibition of DNA synthesis after x or γ irradiation is two to three times less pronounced than in normal cells.7 11 14 16 18
Nineteen of the patients have died, five from infection, and 14 from malignancy. Limited autopsy findings are available: an extensive postmortem examination has been reported for only one patient, a 4 year old child (patient 12), in whom brain weight was extremely reduced. There was no evidence of cerebellar degeneration. The thymus was small, dysplastic, and relatively devoid of lymphoid cells. Limited necropsy data from other patients confirm small brain, thymus dysplasia, or even thymus aplasia.2 13
Registered patients come from 44 families, the majority of eastern European ancestry. Recent genetic studies have provided evidence for a common haplotype of markers present in the families with Eastern European origins, suggesting a common founder effect with regard to mutations causing the disorder. After identification of the gene, mutation detection has revealed a truncating 5 bp deletion, 657–661delACAAA, as the disease causing mutation in these patients (see table 1). Five further truncating mutations were identified in patients with other distinct haplotypes. Four of these mutations are listed in table 1. The fifth, a 842–843insT, was identified in a Mexican patient, who has not yet been registered.
Patients with different genotypes may show the same phenotype, as illustrated by patients 2 and 11 both showing hydronephrosis, by patients 7 and 37 both showing severe T cell immunodeficiency, and by patients 11 and 20 both having developed a lymphoma. On the other hand, patients with the same genotype may vary in phenotypic expression, as illustrated clearly by patient 1 who only has an IgA deficiency and is still quite healthy, while his older brother had agammaglobulinaemia and died young from infection. Other examples are: patient 18 has clinodactyly, patient 19 does not; patient 20 developed a glioma, while patient 21 is still free of malignancy.
The hallmarks of NBS are microcephaly, a typical facial appearance, growth retardation, immunodeficiency accompanied by recurrent infections, chromosomal instability,x ray hypersensitivity, and predisposition to malignancy. Important additional features are skin abnormalities, particularly café au lait spots and vitiligo, and congenital malformations, particularly clinodactyly and syndactyly.
Psychomotor development is usually normal or only mildly to moderately retarded despite severe microcephaly. This is in contrast to the severe mental impairment commonly seen in isolated or autosomal recessive non-syndromal microcephaly. About three quarters of the NBS patients are microcephalic at birth and the remainder become microcephalic within the first year of life. No correlation can be seen between head circumference at birth and mental development. Severe microcephaly at birth may be associated with normal mental development and counterwise.
Life expectancy is reduced because of their tendency to develop malignancies at a relatively young age and sometimes fatal infections.
Remarkably, despite the often severe immunodeficiency, frequently occurring infections rarely lead to severe complications. Moreover, opportunistic infections are very sparse despite the T cell defect, as also experienced in ataxia telangiectasia (AT), which has similar T cell defects.
About 40% of the patients included in the NBS registry have developed a malignancy, predominantly in childhood. Treatment may be difficult, because of hypersensitivity to ionising radiation and radiomimetic drugs, which may have to be avoided or used at reduced dosages.18
The immunological, cytogenetic, and cell biological findings in NBS closely resemble those in AT (see table 3). Therefore NBS has long been considered as an AT variant.19-25 Concerning the clinical aspects, however, NBS is more similar to Bloom syndrome (BS), both of which encompass severe microcephaly with relatively preserved mental development. Unlike AT, neurological features are rare. The facial appearance is different, being small and narrow in BS. NBS and BS lack the increased serum α fetoprotein concentrations of AT. BS shows a characteristic cytogenetic feature, the sister chromatid exchanges, which is not seen in NBS and AT.19-25 29
The specific pathogenesis of the disorder still has to be elucidated, despite recent identification of the gene.26-28 This has had immediate implications for diagnosis and family studies. Studies on the gene product, nibrin, have provided a key to new fundamental knowledge on the molecular mechanism of double strand DNA break repair. Comprehensive sequence comparison of nibrin has revealed two domains in the amino terminal region: a forkhead associated domain (FHA) and a breast cancer carboxy terminal domain (BRCT). Both domains have been found separately in DNA damage responsive cell cycle checkpoint proteins. Carney et al 28provided evidence that nibrin can form complexes with two proteins that play a role in double strand break DNA repair. This suggests that deficiency in nibrin disrupts a common pathway that functions to sense or repair double stranded DNA breaks.
DNA double strand breaks occur often but seldom lead to aberrations in case of a normal functioning repair mechanism. In the case of dysfunction of repair mechanisms, however, serious aberrations can be expected, particularly in tissues with high proliferative capacity. In that light, congenital deformations, immunodeficiency, radiation hypersensitivity, and cancer predisposition are comprehensible.
In DNA repair disorders immunodeficiency, growth retardation, and predisposition to malignancy are common, both in man and in animals with Ku70 and Ku80 deficiency. In Ku70 and Ku80 deficient mutants an abnormal sensitivity to ionising radiation and a severe combined immunodeficiency caused by abnormal V(D)J recombination exist. Immunoglobulin heavy chain rearrangements in an NBS lymphoblastoid cell line have been analysed by DNA sequencing and found to be normal.30 However, quantitative analysis in NBS cells is required to address adequately the role of the hMrell/hrad 50 protein complex in this process.28 As IgM level is nearly always normal in NBS, whereas IgG and IgA concentrations are frequently abnormal, this suggests that the gene may be involved in this process during the isotype switch.
No specific genotype–phenotype relation has been identified. NBS registry patients with the same genotype may display different phenotypes, and patients with different genotypes may express the same phenotype. Defects in sensing and repairing double stranded DNA breaks by nibrin on several specific places of the genome as well as the interplay with other gene products, involved in DNA double strand repair, may be the explanation for this feature.
Finally, specific mutations do not lead to specific clinical features. For example, a specific mutation predisposing to malignancy is not seen.
It is interesting that within the same genotype clinical and laboratory features may differ. Obviously compensating mechanisms play a role. Insight into these mechanisms may offer new approaches for therapeutic interventions.
Members of the International NBS Study Group: JA Hiel MD, CM Weemaes MD PhD, LP van den Heuvel PhD, BG van Engelen MD PhD, FJ Gabreëls MD PhD, DF Smeets PhD, and I van der Burgt MD PhD, Departments of Neurology, Pediatrics and Human Genetics, University Hospital, Nijmegen, Netherlands; KH Chrzanovska PhD, E Bernatowska PhD, M Krajewska-Walasek PhD, M Bialecka PhD, D Abramczuk PhD, H Gregorek PhD, J Michalkiewicz PhD, and D Perek PhD, Departments of Medical Genetics and Pathology, Children's Memorial Health Institute, Warsawa; AT Midro PhD, Department of Clinical Genetics, Medical Academy Bialystok, Poland; E Seemanová PhD, Department of Medical Genetics, Charles University, Prague, Czech Republic; BH Belohradsky PhD and B Solder PhD, Department of Immunology, University Hospital, München; G Barbi, Department of Genetics, University Hospital, Ulm; RD Wegner and K Sperling, Institute of Human Genetics, Charite Humbold University, Berlin, Germany; J Dixon PhD, Central Regional Genetic Services, Wellington Hospital, Wellington South, New Zealand; P Maraschio PhD, Department of Pathology, GL Marseglia MD, Department of Pediatric Sciences, University Hospital, Pavia, Italy; A Green PhD, Department of Clinical Genetics, Addenbrooke's NHS Trust, Cambridge; AM Taylor, Department of Cancer Studies, University of Birmingham Medical School, Birmingham, UK; VM Der Kaloustian MD, The McGill University–Montreal Children's Hospital Research Institute, Montreal, Canada; K Komatsu PhD and S Matsuura PhD, Department of Radiation Biology, Hiroshima University, Hiroshima, Japan; ME Conley PhD, Department of Immunology, St Jude Children's Research Hospital, Memphis; P Concannon, Virginia Mason Research Center and Department of Immunology, University of Washington School of Medicine, Seattle; RA Gatti, Department of Pathology, UCLA School of Medicine, Los Angeles, USA.
The International Nijmegen Breakage Syndrome Study Group J A Hiel C M Weemaes L P van den Heuvel B G van Engelen F J Gabreëls D F Smeets I van der Burgt K H Chrzanovska E Bernatowska M Krajewska-Walasek M Bialecka D Abramczuk H Gregorek J Michalkiewicz D Perek A T Midro E Seemanová B H Belohradsky B Sölder G Barbi R D Wegner K Sperling J Dixon P Maraschio G L Marseglia A Green A M Taylor V M Der Kaloustian K Komatsu S Matsuura M E Conley P Concannon R A Gatti
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