Autoimmune diseases and autoantibodies in pediatric patients and their first-degree relatives with immunoglobulin A deficiency

Abstracts

Introduction:

Clinical manifestations of Immunoglobulin A Deficiency (IgAD) include recur-rent infections, atopy and autoimmune diseases. However, to our knowledge, theconcomitant evaluations of autoimmune diseases and auto antibodies in a cohort of IgADpatients with current age >10 years and their relatives have not been assessed.

Objectives:

To evaluate autoimmune diseases and the presence of auto antibodies in IgADpatients and their first-degree relatives.

Methods:

A cross-sectional study was performed in 34 IgAD patients (current age >10years) and their first-degree relatives. All of them were followed at a tertiary Brazilianprimary immunodeficiency center: 27 children/adolescents and 7 of their first-degree rela-tives with a late diagnosis of IgAD. Autoimmune diseases and autoantibodies (antinuclearantibodies, rheumatoid factor, and anti-thyroglobulin, anti-thyroperoxidase and IgA classanti-endomysial antibodies) were also assessed.

Results:

Autoimmune diseases (n = 14) and/or autoantibodies (n = 10, four of them with iso-lated autoantibodies) were observed in 18/34 (53%) of the patients and their relatives. Themost common autoimmune diseases found were thyroiditis (18%), chronic arthritis (12%)and celiac disease (6%). The most frequent autoantibodies were antinuclear antibodies(2%), anti-thyroglobulin and/or anti-thyroperoxidase (24%). No significant differences wereobserved in the female gender, age at diagnosis and current age in IgAD patients with andwithout autoimmune diseases and/or presence of auto antibodies (p > 0.05). The frequen-cies of primary immunodeficiencies in family, autoimmunity in family, atopy and recurrentinfections were similar in both groups (p > 0.05).

Conclusion:

Autoimmune diseases and auto antibodies were observed in IgAD patients dur-ing follow-up, reinforcing the necessity of a rigorous and continuous follow-up duringadolescence and adulthood.

IgA deficiency; Autoimmunity; Autoantibodies; Thyroiditis

Introdução:

As manifestações clínicas da deficiência de imunoglobulina A (DIgA) incluem infecções recorrentes, atopia e doenças autoimunes. No entanto, para o nosso conhecimento, as avaliações concomitantes de doenças autoimunes e autoanticorpos em uma coorte de pacientes com DIgA com idade atual > 10 anos e seus parentes não foram feitas.

Objetivos:

Avaliar doenças autoimunes e presença de autoanticorpos em pacientes com DIgA e seus parentes de primeiro grau.

Métodos:

Estudo transversal feito em 34 pacientes com DIgA (idade atual > 10 anos) e em seus parentes de primeiro grau. Todos foram acompanhados em um centro terciário brasileiro para imunodeficiência primária: 27 crianças/adolescentes e sete de seus parentes de primeiro grau com diagnóstico tardio de DIgA. Doenças autoimunes e autoanticorpos (anticorpos antinucleares, fator reumatoide e antitireoglobulina, antitiroperoxidase e anticorpos antiendomísio da classe IgA) também foram avaliadas.

Resultados:

Doenças autoimunes (n = 14) e/ou autoanticorpos (n = 10, quatro deles com autoanticorpos isolados) foram observadas em 18/34 (53%) dos pacientes e seus parentes. As doenças autoimunes mais comuns encontradas foram tireoidite (18%), artrite crônica (12%) e doença celíaca (6%). Os autoanticorpos mais frequentes foram anticorpos antinucleares (2%), antitireoglobulina e/ou antitireoperoxidase (24%). Nenhuma diferença significativa foi observada no sexo feminino, idade no momento do diagnóstico e idade atual em pacientes com DIgA com e sem doenças autoimunes e/ou presença de autoanticorpos (p > 0,05). As frequências de imunodeficiência de primárias na família, autoimunidade em família, atopia e infecções recorrentes foram semelhantes em ambos os grupos (p> 0,05).

Conclusão:

Doenças autoimunes e autoanticorpos foram observadas em pacientes com DIgA durante o acompanhamento, o que reforça a necessidade de um acompanhamento rigoroso e contínuo durante a adolescência e a idade adulta.

Deficiência de IgA; Autoimunidade; Autoanticorpos; Tireoidite

Introduction

IgA deficiency (IgAD) is the most frequent primary immunodeficiency (PID). It is a defect which is caused due to terminal B lymphocyte differentiation, resulting in an insufficient production of serum and secretory IgA (SIgA).^1–4 SIgA has some protective functions on mucosa, neutralizing microorganisms and proteins.^5–8

The clinical manifestations of IgAD patients range from asymptomatic to recurrent infections, allergic symptoms and autoimmune diseases,⁹ with an increased autoantibodies production.¹⁰ Of note, autoimmune diseases occur in 7–36% of IgAD patients and autoantibodies are observed in more than 40% of the patients.^10,11 The prevalence of IgAD is 1–4% in systemic lupus erythematosus (SLE) patients,¹⁰ 2–4% in rheumatoid arthritis (RA)¹⁰ and 2.6% in celiac disease (CD).¹² Furthermore, autoimmune diseases are frequently reported in relatives of IgAD patients. Among the first-degree relatives of IgAD patients, 10% had autoimmune diseases compared to 5% in general population.^11–15

However, to our knowledge, the concomitant evaluation of autoimmune diseases and auto antibodies in a cohort of IgAD patients with current age greater than 10 years and their relatives with IgAD has not been studied.

Therefore, the aim of this study was to evaluate the occurrence of autoimmune diseases and auto antibodies in a cohort of IgAD patients with current age greater than 10 years and their respective first-degree relatives followed at a tertiary Brazilian reference center for pediatric PID.

Patients and methods

We selected 126 IgAD patients followed at a Brazilian pediatric reference center for PID in the last 30 consecutive years. Ninety-two of the IgAD patients who were diagnosed at childhood had current age lower than 10 years and were excluded from this study. IgA assessment was systematically performed in all first-degree relatives that presented any symptoms or signals of recurrent infectious and autoimmune diseases, and IgAD diagnosis was established in 7/62 (11%) of first-degree relatives. Therefore, a cross-sectional study was carried out in 34 IgAD patients: 27 IgAD patients (current age greater than 10 years) and their 7 first-degree relatives with a late diagnosis of IgAD. The study was approved by the Ethical Committee and the written informed consent was obtained from all participants.

A systematic clinical evaluation was performed and included the assessments of various autoimmune disorders, recurrent infectious episodes, atopic manifestations and current or past neoplasia in the IgAD patients and their respective families. IgAD was diagnosed according to Pan-American Group for Immunodeficiency and European Society for Immunodeficiency with exclusion of secondary IgAD.¹⁶

Juvenile idiopathic arthritis (JIA) was diagnosed according to International League of Associations for Rheumatology (ILAR) criteria.¹⁷ Childhood-onset SLE (c-SLE) was diagnosed according to American College of Rheumatology criteria.¹⁸ Ankylosing spondylitis (AS) was defined according to New York criteria.¹⁹ Hypothyroidism was defined as reduced free thyroxine (T4) and elevated thyroid stimulating hormone (TSH) levels. Subclinical hypothyroidism was diagnosed as elevated TSH associated with normal T4. The presence of antithyroid antibodies was required to characterize autoimmune thyroiditis.²⁰ CD was characterized by at least four of the following findings: clinical manifestations (chronic diarrhea, stunting and/or iron deficiency anemia), positivity for CD IgA antibodies, HLA-DQ2 or DQ8 genotype, small intestine biopsy compatible with celiac enteropathy and response to gluten-free diet.²¹

Blood samples were collected from all IgAD patients and their relatives. Serum immunoglobulins (IgA, IgM and IgG) were measured by nephelometry (Behring Laser Nephelometer, USA) and the results were confirmed if they were positive. All patient samples were screened for antinuclear antibodies (ANA) by indirect immunofluorescence with HEp-2 cells (Euroimmun AG, Germany). Dilutions ≥1:80 were defined as positive. Rheumatoid factor (RF) was detected by immunonephelometry (Laborclin, Pinhais, Parana, Brazil, and reference value >8 IU/mL). The serum levels of TSH, free thyroxine (free T4), thyroglobulin, anti-thyroglobulin antibodies (anti-TG) and anti-thyroperoxidase antibodies (anti-TPO) were also determined. TSH and free T4 were measured by immunometric assays (Auto Delphia, Wallac Oy, Finland). The anti-TG and anti-TPO concentrations were determined using fluorescence enzymatic immunoassays (Auto Delphia, Wallac Oy, Finland; reference value >35 IU/mL). IgA class anti-endomysial (EMA) antibody of IgA isotype was evaluated by indirect immunofluorescence, using the umbilical cord as substrate (Dako, Copenhagen, Denmark; reference value >1:10).

Statistical analysis

Results were presented as mean ± standard deviation or median (range) for continuous and number (%) for categorical variables. Data were compared by t- test or Mann–Whitney test for continuous variables. Differences of categorical variables were assessed by Fisher's exact test. In all of the statistical tests, the level of significance was set at 5% (p < 0.05).

Results

Autoimmune diseases (n = 14) and/or autoantibodies (n = 10; four of them with isolated auto antibodies) were observed in 18/34 (53%) of patients and their relatives.

The most common autoimmune disorder was thyroid autoimmune disease in 6 (18%) of the IgAD subjects: hypothyroidism ( n = 2), subclinical hypothyroidism (n = 2) and hyperthyroidism (n = 2). Four patients had chronic arthritis: three patients had JIA (persistent oligoarticular subtype) and the fourth patient had AS during adulthood. One patient had isolated haemolytic autoimmune anemia and the other patient had c-SLE with autoimmune thrombocytopenia. One patient and his sister had CD with an improvement after gluten-free diet.

Table 1 includes demographic data and clinical features in 34 IgAD patients and their first-degree relatives according to the presence of autoimmune diseases and/or autoantibodies. No significant differences were observed in the female gender, age at diagnosis and current age in IgAD patients with and without the presence of autoimmune diseases and/or of autoantibodies (p > 0.05). Likewise, the frequencies of PID and the presence of autoimmune diseases in family, atopy and recurrent infections were similar in both groups (p > 0.05) (Table 1). Upper respiratory tract infections were the most common findings, especially sinusitis (68%) and acute media otitis (58%).

Table 1
Demographic data and clinical features in 34 IgA deficiency (IgAD) patients according to the presence of autoimmune diseases and/or auto-antibodies.

Variables	Autoimmune diseases and/or auto antibodies	p
	With ( n = 18)	Without ( n = 16)
Demographic data
Female	10 (56)	9 (56)	1.0
Age at IgAD diagnosis, yrs	8.9 (4–34)	13.7 (4–52)	0.25
Current age, yrs	19.8 (10–34)	19.6 (10–52)	0.98
Clinical features
PID in family	9 (50)	8 (50)	1.0
Autoimmune diseases in family	2 (11)	3 (19)	0.65
Atopy	11 (61)	11 (69)	0.73
Recurrent infections	15 (83)	16 (100)	0.23

• Results are presented in n (%) or median (range); PID, primary immunodeficiency.

Autoantibodies were observed in 10/34 (29%) of patients and their first-degree relatives (four of them had autoantibodies without autoimmune diseases). High serum titers of anti-TG and anti-TPO antibodies were observed in seven and five patients, respectively. One first-degree relative with CD had an ANA titer of 1:160 and one patient with c-SLE had an ANA titer of 1:320. In the patients without clinical autoimmune manifestations, two of them had positivity for both anti-TG and anti-TPO antibodies and the other two patients had only ANA positivity. Dense fine speckled patterns were observed in ANA-HEp-2 cells of all patients. RF was absence in all IgAD subjects.

Demographic data and clinical features in 34 IgAD patients according to the presence of auto antibodies are shown in Table 2. Female gender, age at diagnosis and current age were similar in IgAD patients with and without the presence of auto antibodies (p > 0.05). PID and autoimmune diseases in family, atopy and recurrent infections were also comparable in both groups (p > 0.05, Table 2).

Table 2
Demographic data and clinical features in 34 IgA deficiency (IgAD) patients according to presence of autoantibodies.

Variables	Autoantibodies	p
	With (n = 10)	Without (n = 24)
Demographic data
Female	7 (70)	12 (50)	0.45
Age at IgAD diagnosis, yrs	8.6 (4–30)	12.2 (4–52)	0.42
Current age, yrs	17.4 (12–30)	20.8 (10–52)	0.35
Clinical features
PID in family	5 (50)	12 (50)	1.0
Autoimmune diseases in family	2 (20)	3 (13)	0.62
Atopy	6 (60)	16 (67)	0.71
Recurrent infections	9 (90)	22 (92)	1.0

• Results are presented in n (%) or median (range); PID, primary immunodeficiency.

Discussion

Antibody deficiencies are the most common PID in our University Hospital, particularly IgAD.^22,23 The present study showed a high prevalence of autoimmune diseases and autoantibodies in IgAD patients followed-up at a tertiary teaching center.

Of note, various clinical manifestations may occur in IgAD patients, ranging from asymptomatic to recurrent infections, allergic symptoms and autoimmune diseases. The clinical spectrum probably depends on the association with antibody deficiencies, such as IgG2 subclass deficiency, specific antibody deficiency, mannose-binding lectin deficiency or common variable immunodeficiency.²⁴ Other possible pathogenesis is a compensatory mechanism, such as high salivary IgM and IgG.²⁵ In the present study, none of the IgAD patients had any other PID.

Autoimmune diseases occur more frequently in IgAD patients compared to healthy populations,^12,22 with the possibility of autoantibodies production, even without autoimmune clinical manifestations,^10,13 as observed herein. Both systemic and organ-specific autoimmune diseases have been described in association with IgAD¹³ and the main autoimmune disorders associated with this immunodeficiency were: hypothyroidism, CD and rheumatic diseases.^9–13,26 Recently, IgAD has also been evidenced in 4% of our c-SLE population.²⁷

Additionally, hematologic autoimmune disorders are very frequent in PID patients, especially antibody deficiencies, such as common variable immunodeficiency and IgAD, particularly idiopathic thrombocytopenic purpura and autoimmune haemolytic anemia, as evidenced in two of our IgAD patients.²⁸

Importantly, the diagnosis of CD in IgAD patients may be very difficult, since the majority of the tests are based on specific IgA antibody. Therefore, it is important to measure the total serum IgA levels before the CD diagnosis.²⁹

Regarding pathogenesis of autoimmunity in IgAD patients, the absence of IgA on mucosal surfaces induces absorption of many environmental antigens, such as diet proteins, and may provoke cross-reaction with self-antigens.^30,31 Moreover, the inability of defective immune response to handle these antigens may result in a compensatory response, thus leading to tissue damage and autoimmunity.^32,33 In addition, specific haplotypes (HLA-A1, HLA-B8 and HLA-DR3) were also found in IgAD patients associated with autoimmune diseases.³³

Isolated ANA was also observed in our asymptomatic patients. Indeed, a Brazilian study showed that 12.6% of healthy children and adolescents in São Paulo had a positive ANA titer >1/80, without the presence of other autoantibodies. However, in this study IgA levels were not assessed.³⁴

Auto antibodies were observed in 29% of our IgAD patients. This frequency was higher than our pediatric leprosy patients (16%)³⁵ and was lower than our c-SLE, juvenile dermatomyositis³⁶ and RASopathies³⁷ patients, who presented up to 93%, 59% and 52% of auto antibodies, respectively.

Another relevant result of the present study was the identification of IgAD in first-degree relatives, most of them asymptomatic, reinforcing the importance of PID evaluation in family members. Although many relatives had autoimmune diseases and mild infections, these manifestations were neglected by them and by physicians. Therefore, autoimmune diseases and infectious manifestations should be considered in the relatives with IgAD.

This study has limitations. These patients were referred to a tertiary university hospital, some of them with more complex diseases, which may have overestimated the autoimmune disorders in this group. Additionally, infants and children lower than 10 years, which is the most prevalent age group for this kind of PID, were excluded. A healthy control group was also not assessed.

In conclusion, a high prevalence of autoimmune diseases and autoantibodies was observed in IgAD patients, reinforcing the rigorous and continuous follow-up during adolescence and adulthood.

• Fundings
  This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – grants 2008/58238-4 to CAS, MCS and CMAJ), by Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPQ – grant 302724/2011-7 to CAS and 308105/2012-5 to CMAJ), by Federico Foundation to CAS and by Núcleo de Apoio à Pesquisa “Saúde da Criança e do Adolescente” da USP (NAP-CriAd).

BIBLIOGRAFIA

• ¹
  Carneiro-Sampaio M, Liphaus BL, Jesus AA, Silva CA, Oliveira JB, Kiss MH. Understanding systemic lupus erythematosus physiopathology in the light of primary immunodeficiencies. J Clin Immunol. 2008;28:34–41.
• ²
  Carneiro-Sampaio MM, Carbonare SB, Rozentraub RB, De Araujo MN, Ribeiro MA, Porto MH. Frequency of selective IgA deficiency among Brazilian blood donors and healthy pregnant women. Allergol Immunopathol (Madr). 1989;17:213–6.3.
• ³
  Leiva LE, Zelazco M, Oleastro M, Carneiro-Sampaio M,Condino-Neto A, Costa-Carvalho BT, et al. Latin AmericanGroup for Primary Immunodeficiency Diseases, Primaryimmunodeficiency diseases in Latin America: the secondreport of the Lagid registry. J Clin Immunol. 2007;27:101–8.4
• ⁴
  Kralovicova J, Hammarström L, Plebani A, Webster AD, Vorechovsky I. Fine-scale mapping at IGAD1 and genome-wide genetic linkage analysis implicate HLA-DQ/DR as a major susceptibility locus in selective IgA deficiency and common variable immunodeficiency. J Immunol. 2003;170:2765–75.5
• ⁵
  Tomasi TB. The discovery of secretory IgA and the mucosalimmune system. Immunol Today. 1992;13:416–8.
• ⁶
  Mestecky J, McGhee JR, Immunoglobulin A. (IgA): Molecular and cellular interactions involved in IgA biosynthesis and immune response. Adv Immunol. 1987;40:153–245.
• ⁷
  Lamm ME. Current concepts in mucosal immunity IV. How epithelial transport of IgA antibodies relates to host defense. Am J Physiol. 1998;274:614–27.
• ⁸
  Steuer A, McCrea DJ, Colaco CB. Primary Sjogren’s syndrome, ulcerative colitis and selective IgA deficiency. Postgrad Med J. 1996;72:499–500.
• ⁹
  Janzi M, Kull I, Sjöberg R, Wan J, Melén E, Bayat N, et al.Selective IgA deficiency in early life: association to infectionsand allergic diseases during childhood. Clin Immunol.2009;133:78–85.
• ¹⁰
  Liblau RS, Bach JF. Selective IgA deficiency and autoimmunity. Int Arch Allergy Immunol. 1992;99:16–27.
• ¹¹
  Fusaro AE, Fahl K, Cardoso EC, de Brito CA, Jacob CM, Carneiro-Sampaio M, et al. Profile of autoantibodies against phosphorylcholine and cross-reactivity to oxidation-specific neoantigens in selective IgA deficiency with or without autoimmune diseases. J Clin Immunol. 2010;30:872–80.
• ¹²
  Cataldo F, Marino V, Ventura A, Bottaro G, Corazza GR. Prevalence and clinical features of selective immunoglobulin A deficiency in coeliac disease: an Italian multicentre study Italian Society of Paediatric Gastroenterology and Hepatology (Sigep) and Club del Tenue Working Groups on Coeliac Disease. Gut. 1998;42:362–5.
• ¹³
  Arkwright PD, Abinun M, Cant AJ. Autoimmunity in human primary immunodeficiency diseases. Blood. 2002;99:2694–702.
• ¹⁴
  Carneiro-Sampaio MM, Coutinho A. Tolerance and autoimmunity: lessons at the bedside of primary immunodeficiencies. Adv Immunol. 2007;95:51–82.
• ¹⁵
  Leman Yel. Selective IgA deficiency. J Clin Immunol. 2010;30:10–6.
• ¹⁶
  Conley ME, Notarangelo LD, Etzioni A. Diagnostic criteria for primary immunodeficiencies Representing Pagid (Pan-American Group for Immunodeficiency) and Esid (European Society for Immunodeficiencies). Clin Immunol. 1999;93:190–7.
• ¹⁷
  Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J, et al. International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol. 2004;31:390–2.
• ¹⁸
  Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40:1725.
• ¹⁹
  Van Der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis: a proposal for modification of the New York criteria. Arthritis Rheum. 1984;27:361–8.
• ²⁰
  Franklyn JA. Hypothyroidism. Medicine. 2005;33:27–9.
• ²¹
  Catassi C, Fasano A. Celiac disease diagnosis: simple rules are better than complicated algorithms. Am J Med. 2010;123:691–3.
• ²²
  Jacob CM, Pastorino AC, Fahl K, Carneiro-Sampaio M,Monteiro RC. Autoimmunity in IgA deficiency: revisiting therole of IgA as a silent housekeeper. J Clin Immunol.2008;28:56–61.
• ²³
  Carneiro-Sampaio M, Moraes-Vasconcelos D, Kokron CM, Jacob CM, Toledo-Barros M, Dorna MB, et al. Primary immunodeficiency diseases in different age groups: a report on 1,008 cases from a single Brazilian reference center. J Clin Immunol. 2013;33:716–24.
• ²⁴
  Santaella MR, Peredo R, Disdier OM. IgA deficiency: clinical correlates with IgG subclass and mannan-binding lectin deficiencies. P R Health Sci J. 2005;24:107–10.
• ²⁵
  Brandtzaeg P, Karlsson G, Hansson G, Petruson B, Björkander J, Hanson LA. The clinical condition of IgA-deficient patients is related to the proportion of IgD-and IgM-producing cells in their nasal mucosa. Clin Exp Immunol. 1987;67:626–36.
• ²⁶
  Etzioni A. Immunodeficiency and autoimmunity. Autoimmun Rev. 2003;2:364–9.
• ²⁷
  Jesus AA, Liphaus BL, Silva CA, Bando SY, Andrade LE, Coutinho A, et al. Complement and antibody primary immunodeficiency in juvenile systemic lupus erythematosus patients. Lupus. 2011;20:1275–84.
• ²⁸
  Cunningham-Rundles C. Hematologic complications of primary immune deficiencies. Blood Rev. 2002;16:61–4.
• ²⁹
  Sinclair D, Saas M, Turk A, Goble M, Kerr D. Do we need to measure total serum IgA to exclude IgA deficiency in coeliacdisease? J Clin Pathol. 2006;59:736–9.
• ³⁰
  Fairweather D, Kaya Z, Shellan GR, Lawson CM, Rose NR. From infection to autoimmunity. J Autoimmun. 2001;16:341–5.
• ³¹
  Monteiro RC, Van De Winkel JG. IgA Fc receptors. Annu Rev Immunol. 2003;21:177–204.
• ³²
  De Laat PC, Weemaes CM, Gonera R, Van Munster PJ, Bakkeren JA, Stoelinga GB. Clinical manifestations in selective IgA deficiency in childhood. A follow-up report. Acta Paediatr Scand. 1991;80:798–804.
• ³³
  Heikkila MK, Oistinen J, Lohman M, Koskimies S. Increase frequencies of HLA -A1 and B-8 in association with total lack, but not with deficiency of serum IgA. Tissues Antigens. 1984;23:280–3.
• ³⁴
  Hilário MO, Len CA, Roja SC, Terreri MT, Almeida G, Andrade LE. Frequency of antinuclear antibodies in healthy children and adolescents. Clin Pediatr (Phila). 2004;43:637–42.35
• ³⁵
  Neder L, Rondon DA, Cury SS, Silva CA. Musculoskeletal manifestations and autoantibodies in children and adolescents with leprosy. J Pediatr (Rio J). 2014 (in press).
• ³⁶
  Aikawa NE, Jesus AA, Liphaus BL, Silva CA, Carneiro-Sampaio M, Viana VS, et al. Organ-specific autoantibodies and autoimmune diseases in juvenile systemic lupus erythematosus and juvenile dermatomyositis patients. Clin Exp Rheumatol. 2012;30:126–31.
• ³⁷
  Quaio CR, Carvalho JF, da Silva CA, Bueno C, Brasil AS, Pereira AC, et al. Autoimmune disease and multiple autoantibodies in 42 patients with RASopathies. Am J Med Genet A. 2012;158A:1077–82.

Publication Dates

• Publication in this collection
  May-Jun 2015

History

• Received
  23 Jan 2014
• Accepted
  06 Oct 2014