Identification of PiZ gene products in liver tissue by a monoclonal antibody specific for the Z mutant of alpha 1-antitrypsin

Globular inclusions of abnormal alpha 1-antitrypsin (AAT) in the rough endoplasmic reticulum of hepatocytes is a characteristic feature of AAT deficiency of the PiZ phenotype. It is also seen in some rare M-like Pi types (including M-Cagliari) having low plasma AAT levels and M-like mobility on isoelectric focusing. In this report the ability of a monoclonal antibody (ATZ 11) raised against PiZ hepatocytic AAT to identify AAT inclusions by immunohistochemical techniques is evaluated. The antibody was found to specifically and selectively identify the PiZ gene products in hepatocytes, but not M-Cagliari AAT. Application of the method thus allows distinction of PiZ gene carriers from PiM-like subjects in the absence of serum protein analysis.     

A review of α1-antitrypsin deficiency

α(1)-Antitrypsin (AAT) deficiency is an underrecognized genetic condition that affects approximately 1 in 2,000 to 1 in 5,000 individuals and predisposes to liver disease and early-onset emphysema. AAT is mainly produced in the liver and functions to protect the lung against proteolytic damage (e.g., from neutrophil elastase). Among the approximately 120 variant alleles described to date, the Z allele is most commonly responsible for severe deficiency and disease. Z-type AAT molecules polymerize within the hepatocyte, precluding secretion into the blood and causing low serum AAT levels (～ 3-7 μM with normal serum levels of 20-53 μM). A serum AAT level of 11 μM represents the protective threshold value below which the risk of emphysema is believed to increase. In addition to the usual treatments for emphysema, infusion of purified AAT from pooled human plasma-so-called "augmentation therapy"-represents a specific therapy for AAT deficiency and raises serum levels above the protective threshold. Although definitive evidence from randomized controlled trials of augmentation therapy is lacking and therapy is expensive, the available evidence suggests that this approach is safe and can slow the decline of lung function and emphysema progression. Promising novel therapies are under active investigation.     

Up-regulation of miR-34b/c by JNK and FOXO3 protects from liver fibrosis

α1-Antitrypsin (AAT) deficiency is a common genetic disease presenting with lung and liver diseases. AAT deficiency results from pathogenic variants in the SERPINA1 gene encoding AAT and the common mutant Z allele of SERPINA1 encodes for Z α1-antitrypsin (ATZ), a protein forming hepatotoxic polymers retained in the endoplasmic reticulum of hepatocytes. PiZ mice express the human ATZ and are a valuable model to investigate the human liver disease of AAT deficiency. In this study, we investigated differential expression of microRNAs (miRNAs) between PiZ and control mice and found that miR-34b/c was up-regulated and its levels correlated with intrahepatic ATZ. Furthermore, in PiZ mouse livers, we found that Forkhead Box O3 (FOXO3) driving microRNA-34b/c (miR‐34b/c) expression was activated and miR-34b/c expression was dependent upon c-Jun N-terminal kinase (JNK) phosphorylation on Ser⁵⁷⁴. Deletion of miR-34b/c in PiZ mice resulted in early development of liver fibrosis and increased signaling of platelet-derived growth factor (PDGF), a target of miR-34b/c. Activation of FOXO3 and increased miR-34c were confirmed in livers of humans with AAT deficiency. In addition, JNK-activated FOXO3 and miR-34b/c up-regulation were detected in several mouse models of liver fibrosis. This study reveals a pathway involved in liver fibrosis and potentially implicated in both genetic and acquired causes of hepatic fibrosis.

α1-Antitrypsin (AAT) deficiency is an inherited disorder that affects ∼1 in 3,000 individuals and is an important genetic cause of lung and liver disease (1). The most common defect is the Z variant of the SERPINA1 gene, which results in the production of misfolded and polymerogenic Z α1-antitrypsin (ATZ). ATZ-dependent liver disease has a wide spectrum of clinical manifestations ranging from liver insufficiency in newborns to chronic liver disease and hepatocellular carcinoma in adults (2, 3). Because of its misfolding and polymerization, ATZ is unable to efficiently traverse the secretory pathway. Accumulation of ATZ in the endoplasmic reticulum (ER) of hepatocytes has a proteotoxic effect.Expression of microRNAs (miRNAs) is affected in several liver diseases with distinct profiles across diseases with different etiologies (4). Here, we investigated differentially expressed miRNAs in the liver of PiZ mice, a transgenic animal model expressing the human ATZ (5). We then confirmed the most relevant findings in liver samples from patients. Following the identification of an important miRNA cluster involved in liver fibrosis, the upstream molecules affecting its expression, and its effector, we showed this pathway is involved in various murine models of liver fibrosis.     

A fast amplification-reverse hybridization assay kit to detect the most frequent deficient variants in the alpha-1-antitrypsin gene

BACKGROUND: There is worldwide growing awareness of alpha-1-antitrypsin deficiency (AATD), a major hereditary disorder in Caucasians. The gold standard for its laboratory diagnosis is thin-layer isoelectric focusing, which should be performed in reference laboratories. OBJECTIVES: The aim of this study was to check the characteristics of a commercially available amplification-reverse hybridization assay kit in detecting at a molecular level the alpha-1-antitrypsin (AAT) Z and S variants, i.e. the most frequent variants associated with AATD, by comparing its performance with DNA restriction fragment length polymorphism. METHODS: We studied samples from 36 subjects enrolled in the Italian National Registry for Severe Alpha-1-antitrypsin Deficiency. Based on previous plasma isoelectric focusing typing, we selected samples with the following phenotypes: MM (9 samples), MS (9 samples), SZ (3 samples), MZ (11 samples), ZZ (3 samples), and a rare variant (1 sample). DNA was extracted by the standard method. The presence of the AAT Z and S gene variants was determined by the amplification-reverse hybridization test kit, following the manufacturer's instructions, and by the restriction fragment length polymorphism technique, according to established procedures. RESULTS: We found that the identification of the AAT Z and S gene variants obtained by the amplification-reverse hybridization test kit was completely in agreement with that obtained by the restriction fragment length polymorphism technique. CONCLUSIONS: We conclude that the test kit provides a fast, easy and unambiguous identification of Z and S alleles. Because of its transferability to routine laboratories, the test kit may be useful in identifying cases of severe AATD, thus resulting in increasing awareness of this rare disorder.     

Alpha-1 antitrypsin deficiency among Indian children with liver disorders.

AIMS: To determine the frequency of alpha-1 antitrypsin (AAT) deficiency in children with chronic liver disease (CLD) and neonatal cholestasis syndrome (NCS). METHODS: All children with NCS (n=23) or CLD (n=35) attending the Pediatric Gastroenterology Clinic between November 2003 and July 2005 were screened for AAT deficiency using phenotyping through isoelectric focusing of plasma. RESULTS: Of the 58 children studied, 57 had normal PiMM phenotype. One child with CLD had the M1E type of normal variant. None of the patients had the abnormal phenotype PiZZ. CONCLUSION: AAT deficiency is infrequent among children with CLD and NCS in our region.     

Alpha-1-antitrypsin deficiency in Madeira (Portugal): The highest prevalence in the world

Alpha-1-antitrypsin (AAT) deficiency is a common genetic disease which affects both lung and liver. Early diagnosis can help asymptomatic patients to adjust their lifestyle choices in order to reduce the risk of Chronic Obstructive Pulmonary Disease (COPD). The determination of this genetic deficiency prevalence in Madeira Island (Portugal) population is important to clarify susceptibility and define the relevance of performing genetic tests for AAT on individuals at risk for COPD.Two hundred samples of unrelated individuals from Madeira Island were genotyped for the two most common AAT deficiency alleles, PI*S and PI*Z, using Polymerase Chain Reaction-Mediated Site-Directed Mutagenesis.Our results show one of the highest frequencies for both mutations when compared to any already studied population in the world. In fact, PI*S mutation has the highest prevalence (18%), and PI*Z mutation (2.5%) was the third highest worldwide. The frequency of AAT deficiency genotypes in Madeira (PI*ZZ, PI*SS, and PI*SZ) is estimated to be the highest in the world: 41 per 1000.This high prevalence of AAT deficiency on Madeira Island reveals an increased genetic susceptibility to COPD and suggests a routine genetic testing for individuals at risk.     

Z α-1 antitrypsin deficiency and the endoplasmic reticulum stress response

The serine proteinase inhibitor α-1 antitrypsin (AAT) is produced principally by the liver at the rate of 2 g/d. It is secreted into the circulation and provides an antiprotease protective screen throughout the body but most importantly in the lung, where it can neutralise the activity of the serine protease neutrophil elastase. Mutations leading to deficiency in AAT are associated with liver and lung disease. The most notable is the Z AAT mutation, which encodes a misfolded variant of the AAT protein in which the glutamic acid at position 342 is replaced by a lysine. More than 95% of all individuals with AAT deficiency carry at least one Z allele. ZAAT protein is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum (ER) of hepatocytes and other AAT-producing cells. This results in a loss of function associated with decreased circulating and intrapulmonary levels of AAT. However, the misfolded protein acquires a toxic gain of function that impacts on the ER. A major function of the ER is to ensure correct protein folding. ZAAT interferes with this function and promotes ER stress responses and inflammation. Here the signalling pathways activated during ER stress in response to accumulation of ZAAT are described and therapeutic strategies that can potentially relieve ER stress are discussed.     

The discovery of α1-antitrypsin and its role in health and disease

α1-Antitrypsin (AAT) is the archetype member of the serine protease inhibitor (SERPIN) supergene family. The AAT deficiency is most often associated with the Z mutation, which results in abnormal Z AAT folding in the endoplasmic reticulum of hepatocytes during biogenesis. This causes intra-cellular retention of the AAT protein rather than efficient secretion with consequent deficiency of circulating AAT. The reduced serum levels of AAT contribute to the development of chronic obstructive pulmonary disease (COPD) and the accumulation of abnormally folded AAT protein increases risk for liver diseases. In this review we show that with the discovery of AAT deficiency in the early 60s as a genetically determined predisposition to the development of early-onset emphysema, intensive investigations of enzymatic mechanisms that produce lung destruction in COPD were pursued. To date, the role of AAT in other than lung and liver diseases has not been extensively examined. Current findings provide new evidence that, in addition to protease inhibition, AAT expresses anti-inflammatory, immunomodulatory and antimicrobial properties, and highlight the importance of this protein in health and diseases. In this review co-occurrence of several diseases with AAT deficiency is discussed.     

Z and Mmalton‐1‐antitrypsin deficiency‐associated hepatocellular carcinoma: a genetic study

Background: The histological hallmark of α‐1‐antitrypsin deficiency (AATD) is the presence of periodic acid‐Schiff diastase (PASD)‐resistant positive globules in hepatocytes, with a heterogeneous distribution. It is noteworthy that hepatocellular carcinoma (HCC) arises specifically from the AAT‐negative areas but the reason for this remains unclear. Aim: To determine whether the different distribution of AAT globules within neoplastic and non‐neoplastic hepatocytes is the result of a self‐induced correction of the genetic defect. Patients and Methods: Two HCV‐positive patients with AATD‐associated HCC were studied. One patient harboured a compound heterozygous PiSZ genotype whereas the other showed the rarer PiMMmalton in heterozygosity. In both cases, neoplastic hepatocytes appeared globule devoid, while non‐neoplastic hepatocytes showed intracytoplasmic accumulation of PASD‐positive globules. Laser‐assisted microdissection was used to assess a genotype/phenotype correlation in single liver cells from HCC and from non‐neoplastic hepatocytes. Results: Direct sequencing of DNA purified from globule‐devoid and globule‐filled hepatocytes demonstrated that all liver cells carried the same mutant genetic background. Conclusion: Our findings indicate that (i) both variants of HCC arising in AAT deficiency (Z and Mmalton) do not accumulate the mutant protein and (ii) the different phenotypic appearance of hepatocytes is not the result of a retromutation during neoplastic transformation, but other mechanisms should be investigated.     

The Effects of Inflammation on Alpha 1 Antitrypsin Levels in a National Screening Cohort

Alpha 1 Antitrypsin (AAT) is a highly polymorphic serum protein. Several genetic variants are associated with varying degrees of decreased serum levels; however, these levels can rise in response to infection, inflammation, injury and estrogen levels. Although the effect of inflammation is well established, it has never been studied quantitatively with respect to specific genotypes in a large representative sample. Using data from a national AAT deficiency-targeted screening cohort, we evaluated AAT levels of patients with normal and deficiency genotypes in response to inflammation, indicated by elevated serum C-reactive protein (CRP). Additionally, we utilized a regression analysis to adjust for the effect of inflammation for each genotype. Across all stratified genotype groups, increased AAT levels were observed in patients with CRP ≥5 mg/L. Different AAT phenotypes reacted differently in the acute phase; M showed a strong response and Z a reduced reaction. Nevertheless, we discovered that inflammation significantly masked clinically relevant base AAT levels in some PI^*MZ individuals; approximately a quarter of PI^*MZ samples showed signs of inflammation. Median AAT levels (mg/dL) in the presence of inflammation are given for several genotypes; numbers in parentheses are levels from the cohort without inflammation/adjusted levels from the cohort with inflammation using the newly devised algorithm: PI^*MM: 162 (142/140); PI^*MS: 136 (117/115); PI^*MZ: 104 (85/89); PI^*MF: 161 (132/141); PI^*SS: 115 (96/91); PI^*SZ: 66 (54/50). We conclude that simultaneous determinations of CRP and AAT levels, and genotyping are clinically valuable in defining AAT variants and that the effect of inflammation can be adjusted for.     

The prevalence of alpha(1)-antitrypsin deficiency in a representative population sample from Poland

AIM: Severe alpha(1)-antitrypsin (AAT) deficiency is one of the most common genetic disorders in Caucasians. The aim of the present study was to assess an unbiased frequencies of PI*S and PI*Z alleles using genotyping of a representative sample from the general population of Poland. METHODS: A random sample of age- and gender-stratified residents, aged 20 years or older, was drawn from the municipal directory of Kraków, Poland. The two most common deficiency alleles: PI*S and PI*Z were genotyped with qualitative real-time PCR using degenerative dual-labeled allele-specific fluorescent probes. RESULTS: In the total population of 859 adult subjects (mean age: 49.5 years; range: 20-90), 28 heterozygotes MS, 18 heterozygotes MZ and one homozygote S were diagnosed. The frequency of PI*S allele was 17.5 (95% CI: 11.6-23.9) per 1000; and that of PI*Z was 10.5 (95% CI: 5.8-15.7) per 1000. Therefore, the estimated prevalence of inherited severe AAT deficiency (homozygotes Z) in Poland is 1/9110 (95% CI: 1/4057-1/29,727). CONCLUSIONS: In the whole population of Poland comprising 38 millions, one may expect of about 4189 (95% CI: 1284-9406) subjects with severe AAT deficiency. These numbers are high enough to consider genetic testing being introduced into a common clinical practice.     

Detection of alpha-1-antitrypsin PiZ individuals by SSCP and DNA sequencing in formalin-fixed and paraffin-embedded tissue: a comparison with immunohistochemical analysis

BACKGROUND/AIM: The role of alpha-1-antitrypsin (AAT) deficiency in the development of cirrhosis and carcinoma of the liver can be investigated from the analysis of archival biopsy specimens. Immunohistochemistry can visualize the storage of defective protease inhibitor (Pi) variant Z, but does not allow differentation between homozygous and heterozygous patients. The aim of the study was to establish a method for the detection of the PiZ mutation on the gene level. METHODS: Liver biopsy and autopsy samples in which AAT deficiency was detected immunohistochemically by a monoclonal PiZ-antibody were analyzed by single-strand conformational polymorphism (SSCP) to reliably determine hetero- and homozygote carrier state in the absence of blood samples and to confirm the histological diagnosis. The accuracy of SSCP was verified by direct DNA sequencing. RESULTS: Tissue slices (>0.8 cm2) from 29 consecutive cases with immunohistochemically detected PiZ depositions and from ten PiZ-negative control cases were provided for extraction and amplification of DNA. In comparison to wild-type sequence of AAT exon V, all 29 cases showed band shifts on SSCP analysis, with a heterozygous pattern in 28 patients and a homozygous pattern in one patient. DNA sequence analysis revealed the same single-base mutation at position 342 of AAT exon V. CONCLUSIONS: SSCP analysis proved a sensitive and specific technique for the detection of the PiZ mutation at the gene level, which allowed unequivocal differentiation between heterozygous and homozygous PiZ status from paraffin-embedded archival tissue specimens. Besides a use in diagnostic pathology, this technique could be valuable for prenatal diagnosis and population screening purposes.     

A novel alpha1-antitrypsin null variant （PiQ0Milano）nt (PiQ0Milano)

Alpha1-antitrypsin deficiency is an autosomal recessive disease characterized by reduced serum levels of alpha1-antitrypsin(AAT)due to mutations in the SERPINA1 gene causing early onset pulmonary emphysema and,occasionally,chronic liver disease.We report an incidental finding of a novel null AAT allele,Q0Milano,consisting of a 17 nucleotides deletion in exon 3 of SERPINA1 gene,in an Italian child with persistently increased liver enzymes,a mild decrease in circulating AAT levels and without any pulmonary disease.Q0Milano variant results in an unfunctional protein lacking of AAT active site,as the resultant protein is truncated near PiS locus involved in AAT protein stability.     

High prevalence of alpha 1 antitrypsin phenotypes in viral hepatitis B infected patients in Iran.

OBJECTIVE: Hepatitis B virus (HBV) infection is a major global public health problem. Approximately 2 billion people are infected worldwide and more than 350 million of these individuals are chronic carriers of HBV. Approximately 15-40% of infected patients will develop cirrhosis, liver failure, or hepatocellular carcinoma (HCC). Alpha 1 antitrypsin (AAT) deficiency is one of many factors that may be involved in abnormalities such as liver and lung disease, inflammatory joint diseases, and inflammatory eye diseases. In the present study, the role played by AAT in HBV infected individuals is analyzed. METHODS: AAT phenotyping and trypsin inhibitory capacity (TIC) experiments were performed on 281 HBV infected patients who were referred to Tehran and Zahedan Hepatitis Center for a period of 3 years from June 2001 to September 2003. The same tests were performed on 257 individuals who did not suffer from any systemic diseases (control group). The case group was subdivided into three groups: carrier (36.7%), chronic (50.5%), and cirrhotic (12.8%). RESULTS: The results showed that AAT phenotypes, MS, MZ, M(1)Z, and M(1)S, were significantly higher in the HBV group (p<0.01). In addition, there was a significant difference in AAT phenotypes (MS, MZ, and M(1)Z) among inactive carriers and individuals in the chronic and cirrhotic group (p<0.001). CONCLUSIONS: There is a high prevalence of moderate AAT (MS, M(1)S, and MV) and severely deficient (MZ and M(1)Z) phenotypes in Iranian HBV individuals. In addition, AAT deficiency might be a risk factor for infected HBV individuals progressing from the carrier stage to chronic and cirrhotic stages.     

Analysis of Systemic Biomarkers in COPD Patients

The finding that α1‐antitrypsin (AAT) deficiency, PiZZ, a well‐established genetic risk factor for COPD, is related to high levels of circulating AAT polymers, prompted us to measure serum levels of such polymers and selected markers of inflammation in age‐ and gender‐matched patients with stable COPD and control subjects with and without severe AAT deficiency, and to assess their relationship with each other and with the genetic AAT‐variant. We found that COPD individuals (n = 20), independent of AAT‐variant, had significantly higher serum levels of AAT and its polymers, MMP‐9, sICAM‐1, VEGF and sE‐selectin than controls (n = 30). Subjects with PiZZ COPD (n = 10) showed significantly elevated serum levels of AAT‐polymers, sE‐selectin and sICAM‐1, while patients with PiMM COPD (n = 10) showed higher levels of MMP‐9, VEGF, IL‐8 and MCP‐1 than controls. By using factor analysis we were able to split the analysed biomarkers into two independent components: the first containing MMP‐9, MCP‐1, IL‐8 and VEGF and the second—AAT and its polymers and sE‐selectin. The result from the binomial logistic regression showed that 95.2 percent of the control individuals and 94.7 percent of the COPD patients can be correctly classified on the basis of the measured serum biomarkers. These observations highlight the importance of the finding sets of biomolecules, which could offer new strategies for the diagnosis of COPD and may have value for monitoring progression of COPD.     

Absence of alpha-1-antitrypsin (Pi Null Bellingham) and the early onset of emphysema

Background : Alpha-1-antitrypsin is the body's major inhibitor of human neutrophil elastase, a powerful proteolytic enzyme capable of degrading the common tissue components. There are over 70 genetic variants of alpha-1-antitrypsin, with the Z allele being of greatest clinical relevance. Individuals homozygous for this allele (approximately one in 2500 in Caucasians) have low serum alpha-1-antitrypsin levels (10–20% of normal) and are predisposed to emphysema, especially if they smoke. Much rarer are mutations which result in the complete or almost complete absence of alpha-1-antitrypsin in the serum.
Aim : To determine the cause of complete absence of alpha-1-antitrypsin in a patient who at age 27 years had both emphysema and idiopathic cardiomyopathy.
Methods : Molecular biology techniques were used to sequence the alpha-1-antitrypsin gene. Allele specific amplification was used to show the presence of the mutations in other family members.
Results : Investigation showed that the proband was homozygous for the Pi Null Bellingham variant of alpha-1-antitrypsin due to the mutation Lys 217 (AAG) to Stop (TAG). His grandmother was heterozygous for Pi Null Bellingham and the additional rare variant P Lowell, Asp 256 (GAT) to Val (GTT), a variant that also results in alpha-1-antitrypsin deficiency.
Conclusion : Patients with complete absence of alpha-1-antitrypsin develop premature emphysema not having smoked or after only minimal exposure, and much earlier than the more common Pi Z individuals who have the usual form of alpha-1-antitrypsin deficiency.     

Role of alpha‐1 antitrypsin in human health and disease

Alpha‐1 antitrypsin (AAT) deficiency is an under‐recognized hereditary disorder associated with the premature onset of chronic obstructive pulmonary disease, liver cirrhosis in children and adults, and less frequently, relapsing panniculitis, systemic vasculitis and other inflammatory, autoimmune and neoplastic diseases. Severe AAT deficiency mainly affects Caucasian individuals and has its highest prevalence (1 : 2000–1 : 5000 individuals) in Northern, Western and Central Europe. In the USA and Canada, the prevalence is 1: 5000–10 000. Prevalence is five times lower in Latin American countries and is rare or nonexistent in African and Asian individuals. The key to successful diagnosis is by measuring serum AAT, followed by the determination of the phenotype or genotype if low concentrations are found. Case detection allows implementation of genetic counselling and, in selected cases, the application of augmentation therapy. Over the past decade, it has been demonstrated that AAT is a broad‐spectrum anti‐inflammatory, immunomodulatory, anti‐infective and tissue‐repair molecule. These new capacities are promoting an increasing number of clinical studies, new pharmacological formulations, new patent applications and the search for alternative sources of AAT (including transgenic and recombinant AAT) to meet the expected demand for treating a large number of diseases, inside and outside the context of AAT deficiency.     

Diagnosis of alpha-1-antitrypsin deficiency by DNA analysis of children with liver disease

BACKGROUND: Alpha-1-antitrypsin deficiency is a genetic disorder which is transmitted in a co-dominant, autosomal form. Alpha-1-antitrypsin deficiency affects mainly the lungs and the liver leading, in the latter case, to neonatal cholestasis, chronic hepatitis or cirrhosis. A precise diagnosis of Alpha-1-antitrypsin deficiency may be obtained by biochemical or molecular analysis. OBJECTIVE: The purpose of this study was to use DNA analysis to examine the presence of an alpha-1-antitrypsin deficiency in 12 children suspected of having this deficiency and who showed laboratory and clinical characteristics of the disease. PATIENTS AND METHODS: Twelve patients, aged 3 months to 19 years, who had serum alpha-1-antitrypsin levels lower than normal and/or had hepatic disease of undefined etiology were studied. The mutant alleles S and Z of the alpha-1-antitrypsin gene were investigated in the 12 children. Alpha-1-antitrypsin gene organization was analyzed by amplification of genome through the polymerase chain reaction and digestion with the restriction enzymes Xmnl (S allele) and Taq-1 (Z allele). RESULTS: Seven of the 12 patients had chronic liver disease of undefined etiology and the other five patients had low serum levels of alpha-1-antitrypsin as well as a diagnosis of neonatal cholestasis and/or chronic liver disease of undefined etiology. Five of the 12 patients were homozygous for the Z allele (ZZ) and two had the S allele with another allele (*S) different from Z. CONCLUSION: These results show that alpha-1-antitrypsin deficiency is relatively frequent in children with chronic hepatic disease of undefined etiology and/or low alpha-1-antitrypsin levels (41.6%). A correct diagnosis is important for effective clinical follow-up and for genetic counseling.